NANOSTRAWS METHODS AND APPARATUSES

Abstract

Methods and apparatuses for extracting, modifying and returning cells to/from a patient to treat the patient. These apparatuses and methods may use nanostraws to deliver biologically relevant molecules such as DNA, RNA, proteins etc., into the cells rapidly and efficiently. The methods and apparatuses described herein are repeatedly capable of delivering biologically relevant cargo into cells with high cell viability, dosage control, unaffected proliferation or cellular development, and with high efficiency, including in a continuous or semi-continuous manner.

Claims

1. A method of treating a patient, the method comprising: withdrawing fluid containing cells from a patient; applying force to drive the cells against nanostraws within a vessel of a treatment apparatus, wherein the nanostraws are on an inner surface of the vessel; transferring a material through the nanostraws into the cells; and returning the cells to the patient.

2. The method of claim 1, wherein applying the force comprises applying mechanical force.

3. The method of claim 2, wherein the mechanical force comprises moving the nanostraws to contact the cells.

4. The method of claim 2, wherein applying the force comprises centrifuging the cells to drive the cells into contact with the nanostraws.

5. The method of claim 1, further comprising releasing the force on the cells to release the cells.

6. The method of claim 1, wherein withdrawing fluid containing cells from the patient comprises coupling the patient to a continuous-flow apparatus, further wherein returning the cells to the patient comprises returning the cells from the continuous-flow apparatus back into the patient.

7. The method of claim 1, wherein transferring material through the nanostraws to into the cells comprises transferring material for less than 5 minutes.

8. The method of claim 1, wherein transferring materials comprises transferring for less than 1 minute.

9. The method of claim 1, further comprising expanding the cells.

10. The method of claim 1, wherein the transferring material comprises transferring material from an inner diameter of the nanostraws wherein each nanostraw of the nanostraws are hollow and have an inner diameter of between 5 nm-1500 nm and each nanostraw extends between 2 nm and 50 ?m from the inner surface.

11. The method of claim 1, wherein transferring material further comprises applying a pulsed electrical field to drive the material from the nanostraws into an intracellular volume of the cells.

12. The method of claim 1, wherein transferring the material comprises transferring a biologically relevant cargo.

13. The method of claim 12, wherein the biologically relevant cargo comprises a polynucleotide.

14. The method of claim 1, wherein the cells comprise blood cells.

15. The method of claim 1, wherein the steps of withdrawing, placing, applying and transferring are performed continuously over a period of less than 3 hours.

16. A method of treating a patient, the method comprising continuously: withdrawing fluid containing cells from a patient as part of a first circuit coupling the patient to a treatment apparatus; applying mechanical force to drive the cells against a plurality of nanostraws on an inner surface of the treatment apparatus; transferring a cargo material through the nanostraws into the cells comprising applying a pulsed electromagnetic field; and returning the cells to the patient though a second circuit portion coupling the patient to the treatment apparatus.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The novel features of the apparatuses and methods are set forth with particularity in the claims that follow. A better understanding of the features and advantages will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0038] FIG. 1 is a schematic of a prototype apparatus for using nanostraws to deliver molecules into a plurality of cells

[0039] FIG. 2 illustrates examples of cargo that may be delivered by an apparatus such as the ones illustrated herein into a variety of cell types (shown on the right).

[0040] FIG. 3A (FIGS. 3A1-3A5) illustrates example of nanostraws and methods of fabricating them (e.g., by atomic layer deposition and plasma etching) that may be used as described herein.

[0041] FIG. 3B is a schematic of an apparatus including nanostraw for delivering material into a plurality of cells.

[0042] FIG. 3C illustrates an example of cells growing on surface including nanostraws (see profile on right of FIG. 3C); image on the left of FIG. 3C is a florescent stain showing cells.

[0043] FIG. 4 is a schematic of a method of treating a patient using an apparatus including nanostraws in a dialysis-like arrangement.

[0044] FIGS. 5A-5C show a side schematic view of an exemplary apparatus in which pressure (e.g., mechanical force) is used to press the cells onto the nanostraws by moving the nanostraws onto the cells. This variation may be referred to as a French press apparatus.

[0045] FIGS. 5D-5E show another variation of an exemplary apparatus similar to that shown schematically in FIGS. 5A-5C. In FIG. 5D, the bottom of the chamber may include a plurality of openings or pores through which fluid may be driven; these opening may be too small to allow the cells to allow passage of the cells, but the flow of fluid out of the chamber may drive the cells against the bottom allowing contact with the nanostraws, as shown in FIG. 5E, for delivery of cargo, as shown in FIG. 5F.

[0046] FIGS. 6A-6B show another example of a flow-through device in use. In FIG. 6A, a side view schematically shows the layout of the exemplary assembled device. Cells are injected through inlet 2, passing by underneath the nanostraws, and are collected at outlet 2. In FIG. 6B, the same side view shows the device during delivery of a cargo into cells flowing through the device. In FIG. 6B, the increased pressure in the cargo reservoir makes the nanostraw membrane bend, thereby coming in contact with the cells, allowing for intracellular access.

DETAILED DESCRIPTION

[0047] Described herein are methods and apparatuses for delivering a load into cells using nanostraws. Non-limiting examples of nanostraws are provided below and in the figures.

[0048] For example, for nanostraws may have any appropriate dimensions, including diameters of between about 10-1000 nm and lengths of between about 0.1-25 ?m.

[0049] For nanostraws of diameters 10-1000 nm and lengths of 0.1-25 ?m, spacers in the range between 0 and up to 50 times the cell diameter can be used. For spacers larger than 10 times the cell diameter, the nanostraws will not be able to reach the cells for intracellular delivery. A spacer of about half the cell diameter may be suitable for delivery into embryos.

[0050] The nanostraws can be incorporated into a microfluidic setup in which biologically relevant cargo can be delivered into cells (including non-adherent cells). In such a setup, cells would be transported in microfluidic chambers and interact with nanostraws in one or several of the following ways: pressure, applying an electric and/or magnetic field, controlling osmotic and/or concentration gradients, use of surface interactions and/or species-species interactions, physical inducement such as centrifugal, flow, shear effects, and/or mechanical compression. Such a flow-through device would be able to greatly increase the number of cells that can be delivered into while at the same time being a closed system with minimal risk for contamination. In here, we give an example of one such embodiment in which a mechanical compression similar to the French press technique is used.

[0051] For example, nanostraws can have diameters ranging from 10 to 1000 nm and lengths ranging from 0.1-25 ?m. Their density can be between about 104 to 1012 nanostraws/cm.sup.2. U.S. Pat. No. 9,266,725 describes nanostraw geometry, methods of manufacturing nanostraws, and nanostraw compositions.

[0052] Cargo Solution: Buffer in which the cargo should be diluted can cover a wide range of salt concentrations. The solution in which the cargo is diluted can have an ion concentration ranging from 0 to 10 000 mmol/L. For larger cargo such as plasmid and proteins, we have seen the highest delivery efficiency using 0.1?PBS as the buffer to dilute the cargo in, regardless of type and strength of the applied external force, and regardless of cell type, see FIGS. 10a-10f for delivery efficiencies of Cas9 protein into Jurkat cells centrifuged onto 200?1200 nm nanostraws.

[0053] In some variations an electric field may also be applied. For nanostraws of diameters 10-1000 nm and lengths of 0.1-25 ?m, a pulsed electric field can be applied over the nanostraws in order to aid the delivery of cargo into the cells. Pulse length from 1 ?s to is [e.g., around 200 ?s], pulse frequency from 0.01 Hz to 10 000 Hz [e.g., 40 Hz], duration of the applied pulses from 0.01 s to 10 h [e.g., around 40 s to 180 s]. The pulsed field can have any profile, including square. Static fields might also work for some niche applications. The electric field will enhance the transport of charged species through the nanostraws as well as to help permeabilize the cellular membrane just above the nanostraws. A pulse train of 15-25V, for 150-300 ?s, at 30-50 Hz, for 80-120 s duration may be used in some variations. Higher voltage can be used.

[0054] Any of the nanostraw structures described herein may be part of an apparatus (e.g., device, system, etc.), including deices for transfecting or modifying cells. Any of the methods (including user interfaces) described herein may be implemented as software, hardware or firmware, and may be described as a non-transitory computer-readable storage medium storing a set of instructions capable of being executed by a processor (e.g., computer, tablet, smartphone, etc.), that when executed by the processor causes the processor to control perform any of the steps, including but not limited to: displaying, communicating with the user, analyzing, modifying parameters (including timing, frequency, intensity, etc.), determining, alerting, or the like.

[0055] For example, FIG. illustrates one example of an apparatus that may be configured as a dialysis-like treatment apparatus for continuous or semi-continuous treatment of the a patient, removing cells (e.g., blood cells), treating cells and returning cells to the patient. Because these systems may rapidly deliver cargo into cells, they may be operated in a nearly continuous manner or continuous manner. In FIG. 1, the apparatus 100 is configured as a system that includes one or more patient connections 102 forming a circuit connecting patient fluid (with cells) to the rest of the apparatus 101. The circuit includes an inlet pathway portion 111 from the body to the apparatus and a return pathway portion 113 from the apparatus back to the body. Although separate lines are shown in FIG. 1 (which may return to the same body region or a different body region), in some variations a joint or combined line (having two portions) may be used, or multiple lines may be used.

[0056] The apparatus 101 may include a control 107 that may monitor operation of the apparatus, including fluid flow, temperature, force applied to the cells, energy (electromagnetic energy) applied, pH, etc. One or more sensors may be included (flow sensors, temperature sensors, pH sensors, etc.). The apparatus may include one or more chambers 107 for receiving cells and/or fluid, including nanostraws configured to deliver material to the cells. The system may also include one or more filters, concentrators, or the like 105 for separating our cells to be treated.

[0057] In use, the apparatus 100 may be connected to the patient 103 by coupling the inflow portion of the circuit 111 to the patient and the outflow portion of the circuit 113 to the patient and operating the apparatus. The apparatus may be primed first, e.g., so that the patient may receive flow in a continuous manner during treatment. The circuit may be attached to the patient at any appropriate body region (e.g., on a limb, trunk, etc.). FIG. 4 illustrates another schematic drawing of an apparatus (e.g., system), described in greater detail below.

[0058] The apparatuses described herein may therefore be used to modify (e.g., reprogram) cells with minimal perturbation of the cells and may be used to deliver a variety of cargo materials, including combinations of materials. FIG. 2 schematically illustrates types of materials (e.g., nucleotides, antibodies, small molecules/drugs, etc.) that may be delivered using the nanostraws described herein, as well as the types of cells (e.g., blood cells, lymphocytes, macrophages, etc.). The cells may be isolated from the patient's fluid (e.g., blood) and/or may be added to the fluid removed from the patient following isolation separately.

[0059] In general, nanostraws may be metal oxide nanotubes that include a central lumen through which material may be transferred. FIG. 3A illustrates one example of nanostraws (shown in the SEM of FIG. 3A1), and methods of fabricating them (showing steps 3A2-3A5). Nanostraws may be fabricated by, e.g., atomic layer deposition and plasma etching. Cells can be attached onto the nanostraws acutely (e.g., for a few minutes or hours), allowing temporary access without damaging the cells. For example, FIG. 3B schematically illustrates a portion of an apparatus including a chamber having a plurality of nanostraws. The cells 303 are at least temporarily attached to nanostraws extending through a base 305 (e.g., membrane, plate, etc.). A channel 307 is located behind the plate having the nanostraws and may carry fluid with one or more material (e.g., cargo) to be delivered into the cells. Fluid containing the cargo may be flowed into and out of the channel through one or more inlet 309 and outlet 311 ports. One or more pairs of electrodes (not shown) for generating an electromagnetic field may be included to apply electromagnetic energy to help drive the material into the cell from the nanostraws.

[0060] FIG. 3C illustrates one example of cells (shown on the left) transfected with a florescent marker by driving them onto nanostraws (shown schematically on the right); the florescent marker is driven into the cells as shown.

[0061] Another example of a system including nanostraws for delivering materials (e.g., cargo) into cells removed, transformed, and then returned to the patient's body. In FIG. 4 the apparatus includes a fluid circuit showing removal of blood from the patient and separation of the cells. The fluid separated from the cells may then be returned to the body via a return portion of the circuit. The target cells separated from the blood may then be driven against nanostraws including a cargo (e.g., a Chimeric Antibody Receptor Engineered T Cell/T-Cell receptors, CAR-T/TCR) that transforms or modifies the cells, e.g., T-cells, into therapeutic cells, such as improving the ability of T cell receptors to recognize and attack specific antigenic cell antigens by means of genetic modification. And therefore, they are collectively referred to as T cell receptor redirection technology. In this example, CAR-T directly changes one part of TCR into a specific antibody, allowing these modified T-cells to directly attack cancer cells under the guidance of antibodies. The T cell may invade cancerous tissue with its ability to identify cancer cells.

[0062] In FIG. 4, the apparatus may also include a quality control module, in which the cells are monitored and expression may be confirmed. The cells may optionally be allowed to expand or otherwise rest during this period. Once the quality control module confirms that the cells are ready (e.g., have been transformed), the cells may be returned into the body by inserting them back into the return path, as shown.

[0063] Thus, for example, T-cells may be driven onto the nanostraws as described herein, using a force (e.g., a centrifugation at a speed of about 200-2000 g for 2-20 min, e.g., approximately 750 g for 5 min). After the centrifugation, the cargo may be delivered before the cells go off in suspension again, generally before 30 min. Some cell types, such as large plant cells, can wait longer whereas some cell types, such as small T-cells, may be delivered to within 5 min.

[0064] In general, the buffer in which the cells are suspended during the centrifugation can be chosen to be any kind of liquid that will not lyse the cells. More viscous materials can be used to stabilize the cells on the nanostraws for a longer time and reduce the risk of cells going off into solution before the delivery has been performed.

[0065] In some variations, mechanical force may be applied so that nanostraws can be pressed onto cells in order to gain intracellular access, the so called French press embodiment. In this example, illustrated in FIGS. 5A-5C and 5D-5E, a container incorporating a nanostraw membrane 501 in the bottom is filled with a cargo solution 505, FIG. 5A. Unlike the centrifugation-based method described above, since the nanostraws extend out from the container, facing down, cells are placed on a surface surrounded by a thin spacer whose height is chosen to match the height of the cells of interest. The nanostraw container 501 is placed on top of the cells such as to put the nanostraws and the cells in contact. If needed, extra pressure can be applied by adding another fluid on top of the cargo solution. Once a suitable pressure is applied, cargo can be delivered into the cells, including applying the transfer charge (e.g., applying a pulsed electric field).

[0066] When mechanical force is used to drive the cells into the nanostraws in the French press variation, cells may be dispersed on a flat surface surrounded by walls of a defined height. A container consisting of a porous membrane 501 incorporating nanostraws 503 may be extended downwards placed on top of the cells 507. The container may hold the cargo of interest 505 with the option of an extra weight e.g. oil 509 that can provide extra pressure. When pushed towards the cells (as shown in FIG. 5B), the cells will be squeezed, resulting in the nanostraws coming in close proximity to the cells. The nanostraws can then be used for delivery of a cargo into the cells, either by diffusion due to a concentration gradient or by adding external driving forces such as an electric field, centrifugation, liquid flow, etc.

[0067] Another variations is shown in FIGS. 5D-5F. In this example, as above, there is a receptacle for suspended, non-adherent cells 517 and the substrate with nanostraws may be moved into the receptacle; this may displace some of the fluid, which may pass through the base. The base in this example includes openings 521 (channels, pores, etc.) that may pass the fluid (as shown in FIG. 5E) when the substrate and nanostraws are lowered into the base, and may allow the substrate and nanostraws to drive the cells against the base so that they may be supported while the nanostraws contact them in order to pass cargo into the cells.

[0068] In variations in which the nanostraws are driven against the non-adherent cells, the pressure exerted by the nanostraws may be adjusted or controlled in order to control (e.g., tune) their penetration depth. Gene editing tools such as Crispr proteins can be delivered directly through the nanostraws allowing for quick and efficient genetic editing.

[0069] In some variations, a spacer may be used to adjust the depth of the nanostraws advanced into the cells. For nanostraws of diameters 10-1000 nm and lengths of 0.1-25 ?m, spacers in the range between 0 and up to 50 times the cell diameter can be used. For spacers larger than 10 times the cell diameter, the nanostraws will not be able to reach the cells for intracellular delivery. A spacer of about half the cell diameter may be suitable for delivery into embryos. In general, nanostraws can be incorporated into a microfluidic setup in which biologically relevant cargo can be delivered into non-adherent cells. In such a setup, cells may be transported in microfluidic chambers and interact with nanostraws in one or several of the following ways: pressure, applying an electric and/or magnetic field, controlling osmotic and/or concentration gradients, use of surface interactions and/or species-species interactions, physical inducement such as centrifugal, flow, shear effects, and/or mechanical compression. Such a flow-through device would be able to greatly increase the number of cells that can be delivered into while at the same time being a closed system with minimal risk for contamination.

[0070] FIGS. 6A-6B show another example of a portion of a flow-through apparatus similar to that shown in FIG. 3B. In FIG. 6A, a side view schematically shows the layout of the exemplary assembled device. Cells are injected through inlet 2, passing by underneath the nanostraws, and are collected at outlet 2. In FIG. 6B, the same side view shows the device during delivery of a cargo into cells flowing through the device. In FIG. 6B, the increased pressure in the cargo reservoir makes the nanostraw membrane bend, thereby coming in contact with the cells, allowing for intracellular access. For enhanced delivery, an electric field, E, can be applied between the two ITO slides (over the nanostraw membrane and the cells, e.g., between the reservoir 605 and the channel 611 that the cells are flowing through). A working area 619 may include a deflectable substrate along a lateral side of the first flow path. The deflectable substrate may include a plurality of nanostraws 617 extending through the deflectable substrate and into the first flow path, wherein the plurality of nanostraws extend from the flexible substrate (e.g., the plurality of nanostraws may be hollow and have an inner diameter of between 5 nm-1500 nm). The apparatus also include a reservoir 613 in fluid communication with an inside of the plurality of nanostraws, the reservoir configured to hold the biologically relevant cargo. In this example, the deflectable substrate is configured to deflect between a first position (shown in FIG. 6A) and a second position (shown in FIG. 6B), further wherein the plurality of nanostraws extend further into the first flow path in the second position than in the first position to at least partially occlude the first flow path and secure non-adherent cell in the flow path against one or more of the plurality of nanostraws.

[0071] These apparatuses may be configured as open loop, closed loop or semi-open/semi-closed loop systems. A closed loop system may refer to the overall workflow, in which cells 1101 are injected into the device 1103, transformed, and returned in a sterile container 1105. This may reduce user handling, and enable higher numbers of cells to be transfected per unit time. In addition, it may allow the apparatus to be used clinically, where contamination concerns necessitate use of closed-loop devices. For example, the methods and apparatuses described herein may be used with CAR-T cell reprogramming at a local clinic, without need for technicians to manually oversee the transformation process (as schematically illustrated in FIG. 4, above).

[0072] In any of the variations described herein an electric field, and particularly a varying electric field, may be used to drive cargo into the cell and/or open the cell to the nanostraw. For example, for nanostraws of diameters 10-1000 nm and lengths of 0.1-50 ?m, a pulsed electric field can be applied over the nanostraws in order to aid the delivery of cargo into the cells. Pulse length from 1 ?s to 1 s, (e.g., between about 10 ?s and 500 ?s, between about 50 ?s and about 500 ?s, between about 150 ?s and about 400 ?s, about 200 ?s, etc.) and pulse frequency from 0.01 Hz to 100,000 Hz (e.g., between about 0.1 Hz and about 50 kHz, between about 1 Hz and about 1 kHz, between about 10 Hz and about 100 Hz, about 10 Hz, about 20 Hz, about 30 Hz, about 40 Hz, etc.), a duration of the applied pulses from about 0.01 sec to 10 h (e.g., about 0.1 sec to about 1 hour, about 1 sec to about 5 minutes, about 10 sec to about 300 sec, between about 40 sec to about 180 sec, etc.). The pulsed field can have any profile, including square/rectangular, sinusoidal, triangular, etc. Static fields might also work for some applications. The electric field may enhance the transport of charged species through the nanostraws as well as to help permeabilize the cellular membrane just above the nanostraws. The efficiency of the electric field have so far found to be agnostic to the cell types; and exemplary parameters may include a pulse train of 15-25 V, for 150-300 ?s, at 30-50 Hz, for 80-120 sec duration. In some variations, higher voltage can give higher transfection efficiency, but may lower the cell viability.

[0073] Any of the nanostraw structures described herein may be part of an apparatus (e.g., device, system, etc.), including deices for transfecting or modifying cells. Any of the methods (including user interfaces) described herein may be implemented as software, hardware or firmware, and may be described as a non-transitory computer-readable storage medium storing a set of instructions capable of being executed by a processor (e.g., computer, tablet, smartphone, etc.), that when executed by the processor causes the processor to control perform any of the steps, including but not limited to: displaying, communicating with the user, analyzing, modifying parameters (including timing, frequency, intensity, etc.), determining, alerting, or the like.

[0074] When a feature or element is herein referred to as being on another feature or element, it can be directly on the other feature or element or intervening features and/or elements may also be present. In contrast, when a feature or element is referred to as being directly on another feature or element, there are no intervening features or elements present. It will also be understood that, when a feature or element is referred to as being connected, attached or coupled to another feature or element, it can be directly connected, attached or coupled to the other feature or element or intervening features or elements may be present. In contrast, when a feature or element is referred to as being directly connected, directly attached or directly coupled to another feature or element, there are no intervening features or elements present. Although described or shown with respect to one embodiment, the features and elements so described or shown can apply to other embodiments. It will also be appreciated by those of skill in the art that references to a structure or feature that is disposed adjacent another feature may have portions that overlap or underlie the adjacent feature.

[0075] Terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. For example, as used herein, the singular forms a, an and the are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms comprises and/or comprising, when used in this specification, specify the presence of stated features, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and/or groups thereof. As used herein, the term and/or includes any and all combinations of one or more of the associated listed items and may be abbreviated as /.

[0076] Spatially relative terms, such as under, below, lower, over, upper and the like, may be used herein for ease of description to describe one element or feature's relationship to another element(s) or feature(s) as illustrated in the figures. It will be understood that the spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if a device in the figures is inverted, elements described as under or beneath other elements or features would then be oriented over the other elements or features. Thus, the exemplary term under can encompass both an orientation of over and under. The device may be otherwise oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly. Similarly, the terms upwardly, downwardly, vertical, horizontal and the like are used herein for the purpose of explanation only unless specifically indicated otherwise.

[0077] Although the terms first and second may be used herein to describe various features/elements (including steps), these features/elements should not be limited by these terms, unless the context indicates otherwise. These terms may be used to distinguish one feature/element from another feature/element. Thus, a first feature/element discussed below could be termed a second feature/element, and similarly, a second feature/element discussed below could be termed a first feature/element without departing from the teachings of the present invention.

[0078] Throughout this specification and the claims which follow, unless the context requires otherwise, the word comprise, and variations such as comprises and comprising means various components can be co-jointly employed in the methods and articles (e.g., compositions and apparatuses including device and methods). For example, the term comprising will be understood to imply the inclusion of any stated elements or steps but not the exclusion of any other elements or steps.

[0079] In general, any of the apparatuses and methods described herein should be understood to be inclusive, but all or a sub-set of the components and/or steps may alternatively be exclusive, and may be expressed as consisting of or alternatively consisting essentially of the various components, steps, sub-components or sub-steps.

[0080] As used herein in the specification and claims, including as used in the examples and unless otherwise expressly specified, all numbers may be read as if prefaced by the word about or approximately, even if the term does not expressly appear. The phrase about or approximately may be used when describing magnitude and/or position to indicate that the value and/or position described is within a reasonable expected range of values and/or positions. For example, a numeric value may have a value that is +/?0.1% of the stated value (or range of values), +/?1% of the stated value (or range of values), +/?2% of the stated value (or range of values), +/?5% of the stated value (or range of values), +/?10% of the stated value (or range of values), etc. Any numerical values given herein should also be understood to include about or approximately that value, unless the context indicates otherwise. For example, if the value 10 is disclosed, then about 10 is also disclosed. Any numerical range recited herein is intended to include all sub-ranges subsumed therein. It is also understood that when a value is disclosed that less than or equal to the value, greater than or equal to the value and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value X is disclosed the less than or equal to X as well as greater than or equal to X (e.g., where X is a numerical value) is also disclosed. It is also understood that the throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point 10 and a particular data point 15 are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0081] Although various illustrative embodiments are described above, any of a number of changes may be made to various embodiments without departing from the scope of the invention as described by the claims. For example, the order in which various described method steps are performed may often be changed in alternative embodiments, and in other alternative embodiments one or more method steps may be skipped altogether. Optional features of various device and system embodiments may be included in some embodiments and not in others. Therefore, the foregoing description is provided primarily for exemplary purposes and should not be interpreted to limit the scope of the invention as it is set forth in the claims.

[0082] The examples and illustrations included herein show, by way of illustration and not of limitation, specific embodiments in which the subject matter may be practiced. As mentioned, other embodiments may be utilized and derived there from, such that structural and logical substitutions and changes may be made without departing from the scope of this disclosure. Such embodiments of the inventive subject matter may be referred to herein individually or collectively by the term invention merely for convenience and without intending to voluntarily limit the scope of this application to any single invention or inventive concept, if more than one is, in fact, disclosed. Thus, although specific embodiments have been illustrated and described herein, any arrangement calculated to achieve the same purpose may be substituted for the specific embodiments shown. This disclosure is intended to cover any and all adaptations or variations of various embodiments. Combinations of the above embodiments, and other embodiments not specifically described herein, will be apparent to those of skill in the art upon reviewing the above description.