[0001] This application claims the benefit of United States Provisional Patent Application No. 62/980,564 filed on February 24, 2020. These and all other referenced extrinsic materials are incorporated herein by reference in their entirety. Where a definition or use of a term in a reference that is incorporated by reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein is deemed to be controlling.

Field of The Invention

[0002] The field of the invention is activation of platelets.

Background

[0003] The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0004] Platelet-rich plasma (PRP) therapy uses a patient’s own platelets, i.e., the patient's own healing mechanisms, to promote healing of an injured tissue. PRP injections are prepared by taking the patient’s own blood and centrifuging it to concentrate the platelets. These concentrated platelets are then injected back to the patient’s injured or diseased tissue. The injected platelets release growth factors that stimulate and increase the number of stem cells that would repair the injured body.

[0005] Prior work has used laser radiation to activate platelets before injecting the activated platelets back to the patient. For example, GresTier et al. used Eppendorf microtubes (1.7 ml) to contain the platelets to be activated during radiation. See “The effect of green laser light irradiation on whole blood platelets.” Journal of Photochemistry and Photobiology B: Biology 79 (2005) 43-50. All publications identified herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.

[0006] In U.S. Pat. Pub. No. US 2017/0246470 by Meixiong Wu et al., platelets are stored in an incubator to receive light. In U.S. Pat. Pub. No. 2010/0196497 Al, by Susan M.L. LIM, et al., a test tube was used to contain the platelets during radiation (see Fig. 1).

[0007] However, Eppendorf tubes or test tubes are traditional containers not designed to optimize light reflection. The walls of these test tubes are transparent and non-reflective, so the laser energy is lost through the walls.

[0008] Thus, there is still a need for a container that is optically optimized for activating platelets.

Summary of The Invention

[0009] The inventive subject matter provides apparatus, systems and methods in which a mirrored polygonal vessel is provided for irradiation of platelets. Reflective surfaces of the mirrored polygonal vessel are oriented to reflect irradiating energy that passes through a platelet suspension held within it back through the vessel’s interior, thereby increasing the efficiency of irradiation.

[0010] One embodiment of the inventive concept is a polygonal mirrored vessel for irradiating platelets that includes at least three sidewalls positioned at an angle to each other to form a polygon having an interior. At least one sidewall has a reflective area positioned to reflect impinging irradiation towards the interior of the polygon. The vessel is sterile. The polygon can be a triangle, a square, a pentagon, or a hexagon, and can have beveled edges. Two or more of the sidewalls can have a reflective surface. In some embodiments the polygonal mirrored vessel further has a reflective bottom, and can have a top surface. This top surface can include a transparent area. This transparent area can be centrally located relative to the top surface, and can have a cross sectional area of less than 1 cm ² . In some embodiments this transparent energy is an aperture or through-hole that traverses the top surface. In some embodiments this top surface can be configured to open and close the mirrored polygonal vessel (e.g., coupled to an adjacent wall via a hinge or flexible connector). [0011] Another embodiment of the inventive concept is a method of irradiating platelets by instilling the platelets into a polygonal mirrored vessel as described above and directing a beam of irradiating energy towards the polygonal mirrored vessel. In such embodiments the polygonal mirrored vessel can include a transparent area that is transmissive of the irradiating energy, and the beam of irradiating energy can be directed to traverse the transparent area.

[0012] Another embodiment of the inventive concept is a method of irradiating platelets by opening a top surface of a polygonal mirrored vessel as described above and instilling platelets into the polygonal mirrored vessel. The polygonal mirrored vessel is then closed, and a beam of an irradiating energy is directed towards the polygonal mirrored vessel. In some embodiments the polygonal mirrored vessel includes a transparent area that is transmissive of the irradiating energy, and the beam of irradiating energy is directed such that it traverses the transparent area.

[0013] Various objects, features, aspects and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments, along with the accompanying drawing figures in which like numerals represent like components.

Brief Description of The Drawings

[0014] FIG. 1 provides a schematic depiction of a prior art device. As shown, light (14) provided by a laser light source (12) is directed to a test tube (11) containing platelet-rich plasma (10).

[0015] FIG. 2A depicts a vessel of the inventive concept with a triangular cross section.

[0016] FIG. 2B depicts an alternative vessel of the inventive concept with a triangular cross section.

[0017] FIG. 3A depicts a vessel of the inventive concept with a square cross section.

[0018] FIG. 3B depicts an alternative vessel of the inventive concept with a square cross section. [0019] FIG. 4A provides a cross sectional view of the vessel of FIG. 2A.

[0020] FIG. 4B provides a cross sectional view of a vessel of FIG. 2A with beveled edges. [0021] FIG. 4C provides a cross sectional view of the vessel of FIG. 3A.

[0022] FIG. 4D provides a cross sectional view of a vessel of FIG. 3A with beveled edges.

[0023] FIG. 4E provides a cross section of a polygonal vessel of the inventive concept having five sides.

[0024] FIG. 4F provides a cross section of a polygonal vessel of the inventive concept having six sides.

[0025] FIG. 4G provides a cross section of a polygonal vessel of the inventive concept having eight sides.

[0026]

Detailed Description

[0027] The inventive subject matter provides apparatus, systems and methods in which platelets are irradiated in a mirrored (i.e., reflective) polygonal vessel. It should be appreciated that internal reflection within the mirrored polygonal vessel advantageously improves exposure of the platelets to the irradiating energy relative to prior art methods utilizing conventional, nonreflective sample tubes.

[0028] Various objects, features, aspects and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments, along with the accompanying drawing figures in which like numerals represent like components.

[0029] As used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise.

[0030] Unless the context dictates the contrary, all ranges set forth herein should be interpreted as being inclusive of their endpoints, and open-ended ranges should be interpreted to include only commercially practical values. Similarly, all lists of values should be considered as inclusive of intermediate values unless the context indicates the contrary. [0031] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value with a range is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0032] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0033] The following discussion provides many example embodiments of the inventive subject matter. Although each embodiment represents a single combination of inventive elements, the inventive subject matter is considered to include all possible combinations of the disclosed elements. Thus if one embodiment comprises elements A, B, and C, and a second embodiment comprises elements B and D, then the inventive subject matter is also considered to include other remaining combinations of A, B, C, or D, even if not explicitly disclosed.

[0034] The polygonal mirrored vessel has three or more sidewalls positioned at angles to each other to form a polygon, for example, a triangle, a square, a pentagon, a hexagon, and so forth. It is contemplated that the polygon need not to be a perfect polygon. For example, the edges of the polygon can be beveled, i.e., having a smooth transition from one sidewall to another. For another example, the sidewall could be slightly curved. At least one of the inner surfaces of the sidewalls is reflective, so that incident energy can be reflected multiple times within the vessel to activate the platelets. The reflective surfaces can be made of any suitable material, including glass, plastic, or other material that can reflect greater than 50%, 60%, 70%, 80%, 90%, or 95% of incident light at a desired wavelength. In especially preferred embodiments, the vessels are sterilized and for one-time use only.

[0035] The inventive subject matter also provides methods in which platelets are irradiated in a polygonal mirrored vessel. Platelets are stored in the polygonal mirrored vessel having at least one reflective inner surface, and a beam of light (e.g., a laser beam) is directed towards the polygonal mirrored vessel. In some embodiments, one or more sidewall(s) of the vessel is transparent (i.e., transmitting greater than 50%, 60%, 70%, 80%, 90%, or 95% of incident light at a desired wavelength), and the beam of light travels through the transparent sidewall, preferably at a non-normal incident angle (i.e., not perpendicular to the sidewall).

[0036] In some embodiments, the polygonal mirrored vessel comprises a transparent lid, and the beam of light travels through the transparent lid, preferably at a non-normal incident angle (i.e., not perpendicular to the lid). In preferred embodiments, the platelets are separated from other blood components (e.g., red cells and white blood cells), so that the light energy is not absorbed by those cells. For example, platelets can be provided as a suspension of platelets, such as a platelet-rich plasma.

[0037] In preferred embodiments, the polygonal mirrored vessel has a transparent area, for example, on the top surface of the vessel. The transparent area can occupy only a portion (i.e., less than 50%, 40%, 30% 25%, 20%, 15%, 10%, 5%, 2.5%, 1%, 0.5%, 0.25%, or 0.1%) of the top surface, so that the laser beam can be directed into the vessel through the transparent area but difficult to escape from the transparent area.

[0038] In preferred embodiments, the polygonal mirrored has a surface that can open and close, for example, the top surface of the vessel, so that platelets can be added to the polygonal mirrored vessel by opening the top surface, instilling the platelets into the vessel’s interior volume, and then closing the top surface so that the vessel remains airtight and/or sterile during the irradiation process. Ideally, the entire process including adding platelets to the vessel and the irradiation process is operated in a sterile environment (e.g., a biosafety cabinet, laminar flow chamber, or fume hood). [0039] It is also contemplated to use at least one half-silvered or partially-silvered mirror as at least one of the sidewalls. In such embodiments laser or other incident radiation can be applied directly through such a sidewall to impinge on platelets contained within the vessel. Such an embodiment can eliminate the need for illumination through an opening or window in the sidewall. The term "half- silvered mirror" is used generically herein to mean any mirror having an incomplete reflective coating, so that some desired percentage (less than 100%) of incident light is reflected and the remaining is transmitted. Such mirrors are often used in optical instruments and two-way mirrors.

[0040] An example of an embodiment of the inventive concept is shown in FIG. 2A, which depicts a polygonal mirrored vessel has a triangular cross-sectional shape. Sidewalls 210, 220 and 230, and bottom surface 240 are reflective to the interior so that an incident laser beam 201 can be reflected within the internal volume of the vessel (which is depicted as enclosing a suspension of platelets). The top surface 250 is transparent, so that the laser beam 201 can enter the vessel.

[0041] FIG. 2B depicts another embodiment of a polygonal mirrored vessel with a triangular cross section. In this embodiment sidewalls 210 and 220, top surface 250, and bottom surface 240 are reflective on the interior so that a laser beam 201 can be reflected inside the vessel through a sidewall 230.

[0042] FIG. 3A depicts a polygonal mirrored vessel of the inventive concept with a square cross- sectional shape. Sidewalls 310-340, and bottom surface 350 are reflective to the interior so that an impinging laser beam 301 can be reflected within the vessel’s interior. The top surface 360 is transparent, so that the laser beam 301 can enter the vessel. As shown, the top surface 360 can open up, allowing platelets to be added to the vessel, and then be closed to keep the vessel air-tight and sterile. Although laser illumination is shown as provided through the open top of the vessel, it should be appreciated that such illumination can also be provided through the transparent top surface 360 when it is closed (thereby providing a sealed vessel).

[0043] FIG. 3B depicts an alternative embodiment of a polygonal mirrored vessel with a square cross section where sidewalls 310-340, top surface 360, and bottom surface 350 are reflective to the interior so that a laser beam 301 can be reflected within the vessel’s interior. The top surface 360 includes a transparent region or area 361, so that the laser beam 301 can enter the vessel through the transparent area 361. Preferably, the transparent area 361 has a round shape and is smaller than about 1 cm ² in diameter. In some embodiments, the transparent area 361 is made of a glass or a plastic that is transparent to a wavelength emitted by the laser. In other embodiments, the transparent area 361 is an opening or aperture in the top surface 360 that allows the laser beam 301 to enter the vessel. It contemplated that platelets can be added to the vessel through the opening 361 in such embodiments.

[0044] FIG. 4A provides a cross-sectional view of the polygonal mirrored vessel shown in FIG. 2A. FIG. 4B shows a cross-sectional view of another embodiment of the polygonal mirrored vessel in Fig. 2A, with beveled edges.

[0045] FIG. 4C shows a cross-sectional view of the polygonal mirrored vessel in Fig. 3A.

FIG. 4D shows a cross-sectional view of another embodiment of the polygonal mirrored vessel in Fig. 3A with beveled edges.

[0046] FIG. 4E shows a cross-sectional view of a polygonal mirrored vessel having five sidewalls. FIG. 4F shows a cross-sectional view of a polygonal mirrored vessel having six sidewalls. FIG. 4G shows a cross-sectional view of a polygonal mirrored vessel having eight sidewalls.

[0047] Another embodiment of the inventive concept is a method of illuminating platelets using a polygonal mirrored vessel. In such a method, platelets in liquid suspension are placed within the interior volume of a polygonal mirrored vessel as described above. Such a suspension of platelets can be provided by isolating platelets from blood obtained an individual to be treated or from pooled blood (for example by centrifugation and collection of a platelet-rich layer). Alternatively, a platelet suspension can be obtained in the form of platelets obtained by plasmapheresis of the individual to be treated or one or more donors. In some embodiments, a platelet suspension can be provided as a product of cell culture, for example culture of platelet precursor cells or by differentiation of stem cells in culture. Once instilled into the polygonal mirrored vessel, the platelet suspension can be irradiated immediately or stored for a period of time prior to irradiation. In some embodiments one or more pharmaceutically active compounds can be added to the platelet suspension prior to or during irradiation. [0048] Platelets held in suspension within a polygonal mirrored vessel of the inventive concept can be irradiated using any suitable light source capable of producing the desired wavelength at the desired intensity and for the desired period of time. Suitable light sources can be coherent or non-coherent, and can be polarized or non-polarized. Wavelengths used can range from 1 nm to 10,000 nm, and can be applied as a single wavelength, two or more wavelengths in combination, a range of wavelengths (e.g., a range having a span of 10 nm, 25 nm, 50 nm, 100 nm, 200 nm, 500 nm, or 1,000 nm around a central nominal wavelength). In some embodiments platelets can be irradiated with two or more wavelengths or wavelength ranges in a serial fashion the reflective materials utilized in the mirrored polygonal vessels can be selected to reflect the desired wavelength(s) or wavelength range(s) used. Suitable sources of irradiating energy include natural or filtered sunlight, an incandescent source, an LED, a laser, a maser, and an RF emitter. Such sources can be used in combination with conventional optical devices (e.g. mirrors, lenses, filters, diffraction gratings, etc.) in order to direct and/or modify their emission.

[0049] Irradiation of platelets within a mirrored polygonal vessel of the inventive concept can take place for any suitable period of time (e.g., from 0.1 msec to 24 hours, in any interval in between). In some embodiments platelets can be subjected to two or more periods of irradiation, which can be of equal or different lengths of time. Similarly, the intensity and/or wavelength composition of the irradiation can be identical for different periods of irradiation, or can be different for different periods of irradiation. In such embodiments periods of irradiation can be applied in a periodic fashion. Such periods can be patterned, for example as a waveform.

[0050] Following irradiation, treated platelets can be removed from the mirrored polygonal vessel and infused into an individual to be treated (e.g., by infusion or injection into or near a site to be treated). In some embodiments treated platelets can be infused essentially immediately (e.g., within 30 minutes of irradiation) or can be stored prior to infusion. Inventors believe that such irradiated platelets can be effective to treat systemic and/or inflammatory conditions, including muscle, tendon, connective tissue, and soft tissue injuries.

[0051] It should be apparent to those skilled in the art that many more modifications besides those already described are possible without departing from the inventive concepts herein. The inventive subject matter, therefore, is not to be restricted except in the spirit of the appended claims. Moreover, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms “comprises” and “comprising” should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that the referenced elements, components, or steps may be present, or utilized, or combined with other elements, components, or steps that are not expressly referenced. Where the specification claims refers to at least one of something selected from the group consisting of A, B, C .... and N, the text should be interpreted as requiring only one element from the group, not A plus N, or B plus N, etc.