MTAB-TA-Coated Gold Nanorods and Method of Fabrication
11-mercaptoundecyl trimethylammonium bromide gold nanorod (MTAB-GNR) coated with tannic acid (TA), useful for biological cellular imaging. A method of synthesizing tannic acid-coated 11-mercaptoundecyl trimethylammonium bromide gold nanorods (MTAB-TA GNRs) comprising presenting 11-mercaptoundecyl trimethylammonium bromide gold nanorods (MTAB-GNRs) in solution; functionalizing the MTAB-GNRs with tannic acid (TA) added to the MTAB-GNRs, the tannic acid configured to form a coating around the MTAB-GNRs forming MTAB-TA GNRs. The functionalizing step may further comprise adding a sodium chloride solution to the MTAB-TA GNR solution, and/or adding a MOPS buffer to the solution of tannic acid with MTAB-GNRs, and the tannic acid MTAB-GNR solution may be vortexed after each step. The MTAB-TA GNR solution may be centrifuged to separate the MTAB-TA GNRs from residual tannic acid and supernatant, and sterile water may be added to the MTAB-TA GNR solution and centrifuging performed to remove residual tannic acid from the MTAB-TA GNRs.
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The invention described herein may be manufactured and used by or for the Government of the United States for all governmental purposes without the payment of any royalty.
FIELD OF THE INVENTIONThe present invention relates generally to coated gold nanorods and, more particularly, to MTAB-TA-coated gold nanorods and methods of synthesizing them.
BACKGROUND OF THE INVENTIONNanotechnology holds great potential, and nanomaterials are increasingly being developed for use in industrial, military, and consumer products including a vast array of biomedical applications. Gold nanorods (GNRs) are of particular interest due to their unique optical region absorbance, emission, and electronic properties. Based on these unique characteristics, GNRs have been used in a vast array of biomedical applications. However, the combination of responses from GNRs including elicited toxicity, poor cellular uptake, and loss of NIR optical properties due to intracellular aggregation remain obstacles for nanobased biomedical applications. The toxicity of GNRs is known largely to be due to free and surface-associated cetyl trimethylammonium bromide (CTAB), a cationic surfactant used in the aqueous synthesis of GNRs. Two main strategies have been reported to overcome this surfactant's cytotoxicity: replacement by post-synthesis ligand exchange or noncovalent overcoating by chemical cover layering through electrostatic attraction. Unfortunately, some commonly used surface modifications, for example, polyethylene glycol (PEG) functionalization, can significantly lower cellular uptake of the GNRs into cells, reducing their use in biomedical applications. Furthermore, overcoatings can break down in biological environments over time, resulting in surface leaching of the CTAB, so chemical stabilization by overcoating does not guarantee that the CTA B toxicity is completely mitigated.
Previous studies have reported that TA-coated (tannic acid) GNRs (MTAB-TA GNRs) show reduced toxicity, demonstrate a distinctive form of endosomnal uptake, and display a unique intracellular distribution pattern that reduces particle aggregation. Unfortunately, as the CTAB-TA GNR's aspect ratio (AR) increases, so does the toxicity, possibly due to the remaining CTAB.
To lower the toxicity of the GNRs, the removal or exchange of the CTAB from the GNRs may be performed, e.g. with MTAB. GNRs coated with MTAB (11-mercaptoundecyltrimethylammonium bromide) have been synthesized, but limited information is available in terms of their biocompatibility and characterization within biological matrices. However, it was found that 40% of the MTAB GNRs were taken up by the cells, compared to less than 1% of their PEGylated analogs, exceeding the previously-reported GNR uptake values. However, published TEM (transmission electron microscopy) images showed extensive aggregation of intracellular particles (GNRs), causing a loss of their near infrared (NIR) optical priorities. In particular, the intracellular features exhibited close proximity side-by-side assembly of GNRs, which can result in blue shift of plasmon resonance emissions moving the GNRs spectra out of the target NIR “water window” needed for biomedical applications.
Thus there remains a need for methods of synthesizing gold nanorods (GNRs) with enhanced biocompatibility and cellular uptake, while preventing particle aggregation to preserve key NIR optical properties.
SUMMARY OF THE INVENTIONThe present invention overcomes the foregoing problems and other shortcomings, drawbacks, and challenges of gold nanorods (GNRs) with enhanced biocompatibility and cellular uptake, while preventing particle aggregation to preserve key NIR optical properties. While the invention will be described in connection with certain embodiments, it will be understood that the invention is not limited to these embodiments. To the contrary, this invention includes all alternatives, modifications, and equivalents as may be included within the spirit and scope of the present invention.
In a first embodiment of the invention, an 11-mercaptoundecyl trimethylammonium bromide gold nanorod (MTAB-GNR) is coated with tannic acid (TA). This structure provides several advantages, including enhanced biocompatibility and cellular uptake, while preventing particle aggregation and preserving key NIR optical properties.
According to a second embodiment of the invention, a method of synthesizing tannic acid-coated 11-mercaptoundecyl trimethylammonium bromide gold nanorods (MTAB-TA GNRs), comprises presenting one or more 11-mercaptoundecyl trimethylammonium bromide gold nanorods (MTAB-GNRs) in solution; and functionalizing the MTAB-GNRs with tannic acid (TA) added to the MTAB-GNRs in solution, wherein the tannic acid is configured to form a coating around the MTAB-GNRs and MTAB-TA GNRs are formed. TA may spontaneously form a coating, i.e. shell, layer, lattice, matrix, or capsule, around the GNRs. Accordingly, the solution includes MTAB-TA GNRs in addition to any excess TA or MTAB GNRs, if any. This process results in the formation of MTAB-TA GNRs having enhanced biocompatibility and cellular uptake, while preventing particle aggregation to preserve key NIR optical properties, as described above.
According to another embodiment of the invention, the functionalizing step may further comprise vortexing the tannic acid MTAB-GNR solution. Vortexing the solution may suspend and distribute the various components of the solution effectively, which may promote the formation of greater numbers of MTAB-TA GNRs. Vortexing may be performed after each step, as desired. To be clear, the MTAB-TA GNRs are not dissolved in the solution but are merely present in the solution in order to receive one or more components which may be in the solution or introduced in the solution, e.g. TA.
According to a further embodiment of the invention, the functionalizing step may further comprise adding a solution of sodium chloride to the solution of tannic acid with MTAB-GNRs. The sodium chloride helps to reduce the agglomeration of the TA when the buffer is added.
According to another embodiment of the invention, the functionalizing step may further comprise vortexing the tannic acid MTAB-GNR solution. Vortexing the solution may suspend and distribute the various components of the solution effectively, which may promote the formation of greater numbers of MTAB-TA GNRs.
According to a further embodiment of the invention, the functionalizing step may further comprise adding a MOPS buffer (3-(N-morpholino)propanesulfonic acid) to the solution of tannic acid with MTAB-GNRs. The MOPS Buffer raises the pH and helps to stabilize the TA coating around the MTAB-GNR.
According to another embodiment of the invention, the functionalizing step may further comprise vortexing the tannic acid MTAB-GNR solution. Vortexing the solution may suspend and distribute the various components of the solution effectively, which may promote the formation of greater numbers of MTAB-TA GNRs.
According to a further embodiment of the invention, the functionalizing step may further comprise centrifuging the MTAB-TA GNR solution to separate the MTAB-TA GNRs from residual tannic acid and supernatant.
According to another embodiment of the invention, the functionalizing step may further comprise adding sterile water to the MTAB-TA GNR solution and centrifuging to remove residual tannic acid from the MTAB-TA GNRs. This step may be performed several times, as desired, in order to remove excess tannic acid.
According to a further embodiment of the method of synthesizing tannic acid-coated 11-mercaptoundecyl trimethylammonium bromide gold nanorods (MTAB-TA GNRs), the functionalizing step may further comprise adding a solution of sodium chloride to the solution of tannic acid with MTAB-GNRs; adding a MOPS buffer (3-(N-morpholino) propanesulfonic acid) to the solution of tannic acid with MTAB-GNRs; and centrifuging the MTAB-TA GNR solution to separate the MTAB-TA GNRs from residual tannic acid and supernatant.
According to another embodiment of the invention, the functionalizing step may further comprise vortexing the tannic acid MTAB-GNR solution after each addition. Vortexing the solution may suspend and distribute the various components of the solution effectively, which may promote the formation of greater numbers of MTAB-TA GNRs.
According to a further embodiment of the invention, the functionalizing step may further comprise adding sterile water to the MTAB-TA GNR solution and centrifuging to remove residual tannic acid from the MTAB-TA GNRs. The addition of sterile water and centrifuging may be repeated as necessary in order to remove residual tannic acid. This process is advantageous in that it results in the synthesis of MTAB-TA GNRs which are free of residual TA and supernatant, rendering the MTAB-TA GNRs ready for cellular uptake and imaging.
Additional objects, advantages, and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the present invention and, together with a general description of the invention given above, and the detailed description of the embodiments given below, serve to explain the principles of the present invention.
It should be understood that the appended drawings are not necessarily to scale, presenting a somewhat simplified representation of various features illustrative of the basic principles of the invention. The specific design features of the sequence of operations as disclosed herein, including, for example, specific dimensions, orientations, locations, and shapes of various illustrated components, will be determined in part by the particular intended application and use environment. Certain features of the illustrated embodiments have been enlarged or distorted relative to others to facilitate visualization and clear understanding. In particular, thin features may be thickened, for example, for clarity or illustration.
DETAILED DESCRIPTION OF THE INVENTIONThe following examples illustrate particular properties and advantages of some of the embodiments of the present invention. Furthermore, these are examples of reduction to practice of the present invention and confirmation that the principles described in the present invention are therefore valid but should not be construed as in any way limiting the scope of the invention.
One or more 11-mercaptoundecyl trimethylammonium bromide gold nanorods (MTAB-GNR) may be coated with tannic acid (TA), according to the present invention, in order to make tannic acid MTAB gold nanorods (MTAB-TA GNRs). This structure provides several advantages, including enhanced biocompatibility and cellular uptake, while preventing particle aggregation to preserve key NIR optical properties.
According to a second embodiment of the invention, a method of synthesizing tannic acid-coated 11-mercaptoundecyl trimethylammonium bromide gold nanorods (MTAB-TA GNRs), comprises presenting one or more 11-mercaptoundecyl trimethylammonium bromide gold nanorods (MTAB-GNRs) in solution; and functionalizing the MTAB-GNRs with tannic acid (TA) added to the MTAB-GNRs in solution, wherein the tannic acid, e.g. 24 mM solution, is configured to form a coating around the MTAB-GNRs and MTAB-TA GNRs are formed. This process results in the formation of MTAB-TA GNRs having enhanced biocompatibility and cellular uptake, while preventing particle aggregation to preserve key NIR optical properties, as described above.
The functionalizing step may further comprise vortexing the tannic acid MTAB-GNR solution. Vortexing the solution may suspend the various components of the solution effectively, which may promote the formation of greater numbers of MTAB-TA GNRs. The functionalizing step may further comprise adding a solution of sodium chloride. e.g. 1.5 mM solution, to the solution of tannic acid with MTAB-GNRs. The sodium chloride helps to reduce the agglomeration of the TA when the buffer is added. Vortexing the solution may suspend the various components of the solution effectively, which may promote the formation of greater numbers of MTAB-TA GNRs, and vortexing may be performed after each step, if desired.
The functionalizing step may further comprise adding a MOPS buffer (3-(N-morpholino)propanesulfonic acid), e.g. 20 mM. pH 7.4, to the solution of tannic acid with MTAB-GNRs. The MOPS Buffer raises the pH and helps to stabilize the TA coating around the MTAB-GNR.
In addition, or in the alternative, the functionalizing step may further comprise centrifuging, e.g. 8000 g, the MTAB-TA GNR solution to separate the MTAB-TA GNRs from residual tannic acid and supernatant. Sterile water may be added to the MTAB-TA GNR solution and the solution may then be centrifuged to remove residual tannic acid from the MTAB-TA GNRs.
The functionalizing step may further comprise adding a solution of sodium chloride to the solution of tannic acid with MTAB-GNRs; adding a MOPS buffer (3-(N-morpholino) propanesulfonic acid) to the solution of tannic acid with MTAB-GNRs; and centrifuging the MTAB-TA GNR solution to separate the MTAB-TA GNRs from residual tannic acid and supernatant. As above, the functionalizing step may further comprise vortexing the tannic acid MTAB-GNR solution after each addition. Vortexing the solution may suspend the various components of the solution effectively, which may promote the formation of greater numbers of MTAB-TA GNRs.
The functionalizing step may further comprise adding sterile water to the MTAB-TA GNR solution and centrifuging to remove residual tannic acid from the MTAB-TA GNRs. The addition of sterile water and centrifuging may be repeated as necessary in order to remove residual tannic acid. This process is advantageous in that it results in the synthesis of MTAB-TA GNRs which are free of residual TA and supernatant, rendering the MTAB-TA GNRs ready for cellular uptake and imaging.
Tannic acid-coated 11-mercaptoundecyl trimethylammonium bromide (MTAB-TA) gold nanorods (GNRs) (MTAB-TA GNRs) exhibit no significant decrease in either in vitro cell viability or stress activation after exposures to A549 human alveolar epithelial cells. In addition, MTAB-TA GNRs demonstrate a substantial level of cellular uptake beyond that of known prior art GNRs, while displaying a unique intracellular clustering pattern. This clustering pattern significantly reduces intracellular aggregation, which preserves the GNRs NIR optical properties, and which is vital for biomedical imaging applications.
These results demonstrate how surface chemistry modifications may enhance and improve biocompatibility, may allow for higher rate of internalization with low intracellular aggregation of MTAB-TA GNRs, and may identify them as prime candidates for use in nanobased bio-imaging applications.
Materials and Methods Cell CultureThe human lung, A549, cell line (ATCC) was maintained in RPMI 1640 cell culture media (ATCC) supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin-streptomycin. Cells were grown in a humidified incubator controlled at 37° C. and 5% CO2. The same media composition was used for all GNR-exposure procedures.
Gold Nano RodsCTAB, Silica, MTAB, TA, and GNRs were purchased from Nanopartz, Loveland. Colo., USA. CTAB-TA, and PEG GNRs were synthesized as previously reported. Both thiol PEGs (molecular weight 20,000 and 5000) were obtained from Nanocs (Boston, Mass.), and tannic acid (molecular weight 1701.2) was obtained from Sigma Aldrich (St Louis. Mo. USA).
GNR CharacterizationThe purity and spectral signature of the GNRs were analyzed before the experiment, using UV-Vis spectrometry on a Bio TEK Synergy HT™ (Winooski, Vt., USA) instrument. For evaluation of GNR size and morphology, nanoparticles (GNRs) in solution were placed onto a formvar carbon-coated copper TEM grid (Electron Microscopy Sciences) and dried. They were imaged with transmission electron microscopy (TEM) using a Hitachi H-7600 at an accelerating voltage of 120 kV. To assess the surface charge of the GNRs, zeta potential measurements were taken using laser Doppler electrophoresis on a Malvern Zetasizer™, Nano-ZS. Agglomerate sizes of the GNRs in media were determined through dynamic light scattering (DLS), also on a Malvern Zetasizer™ (Malvern Instruments, MA, USA).
Cellular Viability AssessmentA549 cell viability was evaluated using the CellTiter 96® Aqueous One Solution (MTS) (Promega) which monitors mitochondrial function and MultiTox-Glo™ Assay (LCDC) (Promega) which sequentially measures two protease activities; one is a marker of cell viability, and the other is a marker of cytotoxicity. Cells were seeded into a 96-well plate at a concentration of 2×103 cells per well and the following day treated with the stated GNR conditions. After 24 h, the cells viability was determined in accordance with the manufacturer's protocol.
Measurement of ROS GenerationThe intracellular generation of ROS (reactive oxygen species) after GNR exposure was evaluated using CM-H2DCFDA (Invitrogen) which is based on intracellular esterases and oxidation that yield a fluorescent product that is trapped inside the cell. Cells were seeded into a 96-well plate at a concentration of 2×103 cells per well and the following day treated with the stated GNR conditions. After 6 h, the intracellular ROS generation was determined in accordance with the manufacturer's protocol.
Quantification of Intracellular GNRsA total of 1×105 cells/well were seeded on 12-mm diameter glass slides in a 24-well plate in triplicate then dosed with 5 μg/ml, GNRs for 24 h. The cell samples were then washed three times and treated with concentrated aqua regia for a minimum of 20 minutes to ensure full digestion of samples. The samples were then diluted to a final concentration of 3-1% HCl to HNO3, respectively. The intracellular gold concentration was determined through inductively coupled plasma mass spectrometry (ICP-MS) on a Perkin-Elmer ICP-MS 300D instrument (Santa Clara, Calif., USA). ICP-MS was conducted in standard mode with 20 sweeps per reading, at one reading per replicate, and three replicates per sample with a dwell time of 100 ms. A calibration curve was obtained using blank and four gold standard solutions (0 (blank), 2, 50, 100, and 300 μg/L), and the addition of an internal standard (bismuth) was done to ensure that no interferences were occurring.
CytoViva Darkfield ImagingA549 cells were seeded at 1.25×105 cells per chamber on a 2-well chambered slide and grown for 24 h. The following day, the cells were given a dosage of 20 μg/mL GNRs for 24 h. After 24 h, the cells were fixed with 4% paraformaldehyde and incubated with Alexa Fluor® 555-phalloidin for actin staining and DAPI for nuclear staining (invitrogen). The slides were then sealed and imaged using a CytoViva 150 ultraresolution attachment on an Olympus® BX41 microscope (Aetos Technologies. Inc.).
Hyperspectral ImagingA549 cells were seeded at 1.25×105 cells per chamber on a 2-well chambered slide and grown for 24 h and the following day were exposed to GNRs (20 μg/ml). After 24 h, the cells were fixed with 4% paraformaldehyde. The slides were then sealed and imaged using a CytoViva® hyperspectral imaging system (Auburn, Ala., USA). Image capture times and settings remained constant for all samples. Finally, hyperspectral analysis was performed using CytoViva's hyperspectral image analysis software.
Cellular Internalization StudiesA549 cells were seeded in a 6-well plate at 6×105 cells/well for 24 h and then exposed to the stated GNRs (5 and 20 μg/mL) for the indicated duration, washed, and fixed overnight in 2% paraformaldehyde and 2% glutaraldehyde. The cells were then stained with 1% osmium tetroxide, washed, and subsequently dehydrated with ethanol dilutions ranging from 50 to 100%. The cells were then embedded in LR White resin and cured overnight at 60° C. under a vacuum, after which the samples were sectioned using a Leica® EM UC7 Ultramicrotome. Cell sections 70 nm in thickness were placed on a formvar carbon-coated copper TEM grid (Electron Microscopy Sciences) and were imaged. Transmission electron microscopy (TEM) was performed using a Hitachi® H-7600 with an accelerating voltage of 120 kV. Care was taken to ensure full evaluation of each sectioned sample for the well-represented images.
Statistical AnalysisAll experimental results represent a minimum of three independent trials unless otherwise stated. Data are expressed as the mean±the standard error of the mean (SEM). Statistical calculations were performed using SAS™ (Version 9.1) or GraphPad™ Prism (version 5.02, GraphPad Software Inc. La Jolla, Calif., USA) to determine statistical significance at p values of <0.05, <0.01, or <0.001.
Results and Discussion Gold Nanorod CharacterizationGNR characterization was performed to determine their key physicochemical properties and to verify particle uniformity prior to experiments. TEM images demonstrated that GNR sets were uniform in size and morphology (
Since GNRs are known to exhibit toxicity linked to their physiochemical properties, multiple surface chemistries, charges, ARs, and concentrations of surface modifications on toxicity by evaluating membrane integrity and mitochondrial function were examiner.
Next we examined cellular stress by measuring changes in reactive oxygen species (ROS) after cellular exposure to the various GNRs. The 6-h time point was chosen based on maximum ROS response before cell death. A549 cells showed no significant increase in ROS levels after exposure to MTAB GNRs and MTAB-TA GNRs, even at four times the treatment concentration of the CTAB GNRs that had significantly increased ROS levels (see
In view of their biocompatibility based on these initial findings and their longitudinal SPR peaks in the NIR “water window,” we further explored the cellular association and in vitro hyperspectral signature of MTAB AR 4 GNRs with and without a TA coating.
Cellular Association and In Vitro Hyperspectral SignatureNext, we performed in vitro hyperspectral imaging (HSI) microscopy to investigate the GNR's optical properties after cellular association. HSI combines the use of darkfield microscopy and spectroscopy for the measurement of the reflectance spectrum at individual pixels in a micrograph. HSI analysis has been used successfully for characterizing gold nanoparticle aggregation, protein adsorption, and cell uptake in biological/cellular environments. This in vitro analysis of MTAB GNRs and MTAB-TA GNRs is critical, as many studies have shown that biological/cellular environments can alter the optical properties of GNRs and other nanomaterials. In this case. HSI analysis demonstrated that both GNRs had a strong association to A549 cells and appeared to indicate clustering of both MTAB GNRs and MTAB-TA GNRs (
In addition, the MTAB GNRs displayed a vast array of colors compared to the primarily white appearance of the MTAB GNRs, indicating a shift out of the NIR and into the visible spectra. Further, MTAB-TA GNRs have a more uniform clustering that allows for easier identification and delineation of the GNR clusters. Next, the reflectance spectrum of individual GNR clusters were measured and compiled to create a hyperspectral profile for both MTAB GNRs and MTAB-TA GNRs, as depicted in
In addition. MTAB-TA GNRs demonstrated a greater than 2.5-fold increase in scattering intensity over the MTAB GNRs after cellular association. Together, these results suggest that the MTAB-TA GNR form uniform GNR clusters that may be better able to preserve their NIR optical properties after cellular uptake. This finding may be significant for nanobased bio-imaging and therapeutic applications, because a strong spectra profile in the NIR “water window” alter exposure to biological/cellular environments is required for optimum efficacy in biomedical applications.
One possible explanation of the differences in hyperspectral signatures between MTAB GNRs and MTAB-TA GNRs may be due to differences in GNR uptake. Another possible explanation could be due to differences in aggregation states after cellular association and/or internalization. To test these possibilities, we set out to examine MTAB GNRs' and MTAB-TA GNRs' uptake by inductively coupled plasma mass spectrometry (ICP-MS) and intracellular aggregation states by TEM.
Quantification and Visualization of Cellular UptakeQuantification of cellular uptake in the MTAB GNRs and MTAB-TA GNRs (5 μg/ml) was determined by ICP-MS. Both MTAB GNRs and MTAB-TA GNRs exhibit a high level (44 and 41% of treatment, respectively) of cellular uptake (
The distribution of MTAB GNRs and MTAB-TA GNRs AR4 (5 μg/ml) within the cell was observed using TEM (
Since the concentration of nanoparticles, e.g. GNRs, can influence nanoparticle aggregation, cellular patterning was studied at 20 μg/ml.
Next, we analyzed the GNRs clusters/groupings using ImageJ software (
When designing a gold nanorod for nanobased bioimaging applications, three problems are often difficult to overcome: (1) toxicity, (2) poor cellular uptake, and (3) loss of NIR optical properties due to particle aggregation in biological environments. In this invention, the GNR surface chemistry was modified by replacing CTAB with MTAB and overcoating with TA. This created a novel GNR (MTAB-TA GNR) that forms unique clusters, and exhibits no decrease in cellular viability, no indication of cellular stress, and no alteration of cell morphology, confirming its enhanced biocompatibility, as illustrated in
While the present invention has been illustrated by a description of one or more embodiments thereof and while these embodiments have been described in considerable detail, they are not intended to restrict or in any way limit the scope of the appended claims to such detail. Additional advantages and modifications will readily appear to those skilled in the art. The invention in its broader aspects is therefore not limited to the specific details, representative apparatus and method, and illustrative examples shown and described. Accordingly, departures may be made from such details without departing from the scope of the general inventive concept.
Claims
1. An 11-mercaptoundecyl trimethylammonium bromide gold nanorod (MTAB-GNR) coated with tannic acid (TA).
2-12.
Type: Application
Filed: Dec 15, 2016
Publication Date: Jun 21, 2018
Applicant: Government of the United States, as represented by the Secretary of the Air Force (Wright-Patterson AFB, OH)
Inventors: Anthony B. Polito (Springboro, OH), Saber M. Hussain (Beavercreek, OH)
Application Number: 15/379,581