WO2025091035A1

WO2025091035A1 - Embedding force-sensing microbeads in blood clots to report platelet functions

Info

Publication number: WO2025091035A1
Application number: PCT/US2024/053270
Authority: WO
Inventors: Xuefeng Wang
Original assignee: University of Cincinnati
Current assignee: University of Cincinnati
Priority date: 2023-10-27
Filing date: 2024-10-28
Publication date: 2025-05-01
Anticipated expiration: 2026-04-27

Abstract

A method of determining platelet aggregation forces is provided. The method involves embedding tension sensor-coated microbeads in platelet clots and observing fluorescent signals from the microbeads that correspond to contractile forces exerted on the tension sensor-coated microbeads to report platelet aggregation forces. In one embodiment, the platelet aggregation forces are used as biomechanical markers to evaluate platelet functions. In another embodiment, the fluorescent signals can be read directly by a plate reader.

Description

EMBEDDING FORCE-SENSING MICROBEADS IN BLOOD CLOTS TO REPORT PLATELET FUNCTIONS

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application Serial No. 63/546,023, filed October 27, 2023, which application is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

[0002] The present invention relates to methods of diagnosing platelet disorders.

BACKGROUND OF THE INVENTION

[0003] Platelets, also known as thrombocytes, are minute blood cells essential for the blood clotting process. Platelet disorders in patients can have serious consequences, including increased bleeding risks, heightened clotting tendencies (thrombocytosis), susceptibility to infections, and potential organ damage. Hematologists frequently employ platelet functional tests to evaluate the efficacy of a patient's platelets. However, current methodologies, such as platelet aggregometry and flow cytometry, primarily examine platelet- secreted chemicals or measure changes in absorbance caused by platelet aggregation. These methods suffer from a notable rate of falsenegative or false-positive results. Furthermore, they typically demand a significant blood volume (>5 mL), necessitating venous blood draw. Overall, there is a high demand for a convenient, reliable, and rapid platelet examination technique that requires only a small blood sample.

SUMMARY OF THE INVENTION

[0004] Certain exemplary aspects of the invention are set forth below. It should be understood that these aspects are presented merely to provide the reader with a brief summary of certain forms the invention might take and that these aspects are not intended to limit the scope of the invention. Indeed, the invention may encompass a variety of aspects that may not be explicitly set forth below. [0005] In one aspect of the present invention, a method of determining platelet aggregation forces is provided. The method involves embedding tension sensor-coated microbeads in platelet clots and observing fluorescent signals from the microbeads that correspond to contractile forces exerted on the tension sensor-coated microbeads to report platelet aggregation forces. Tn one embodiment, the platelet aggregation forces are used as biomechanical markers to evaluate platelet functions. In another embodiment, the fluorescent signals can be read directly by a plate reader. In one embodiment, platelet aggregation forces are determined using about 10 pL or less of a subject’s blood.

[0006] In another embodiment, platelet aggregation forces are determined using 5-10 minutes of assay time. In one embodiment, the tension sensor is an Integrative Tension Sensor (ITS). In another embodiment, the microbeads comprise a material selected from the group consisting of polystyrene and polyacrylamide. In one embodiment, the microbeads have a diameter of 3-5 pm. In another embodiment, the tension sensors are grafted to the microbeads through streptavidinbiotin interaction. In one embodiment, the microbeads are used at a ratio of microbeads to platelets in the range of 1 : 10 to 1 : 1000.

[0007] In another aspect of the present invention, a tension sensor-coated microbead is provided. The microbead is a sphere of polystyrene having a sphere surface, and tension sensors are grafted to the sphere surface. In one embodiment, the tension sensor is an Integrative Tension Sensor (ITS). In another embodiment, each microbead is grafted with from 10,000 to 100,000 ITS molecules.

[0008] In another aspect of the present invention, a tension sensor-coated microbead is provided. The microbead is a sphere of polyacrylamide having a sphere surface, and tension sensors are grafted to the sphere surface. In one embodiment, the tension sensor is an Integrative Tension Sensor (ITS). In another embodiment, each microbead is grafted with from 10,000 to 100,000 ITS molecules.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The foregoing summary, as well as the following detailed description of preferred embodiments of the application, will be better understood when read in conjunction with the appended drawings.

[0010] FIG. 1A is a schematic showing a double-stranded DNA-based tension sensor, decorated by a quencher, a dye (Atto647N or other dyes) and a ligand (RGD peptide), that attracts platelet adhesion.

[0011] FIG. IB is a schematic showing the process of acquiring platelet adhesive force patterns. [0012] FIG. 1C a series of typical platelet adhesive force images shown after platelet plating on ITS surfaces after 1-10 minutes.

[0013] FIG. 2 is a schematic showing ITS-coated microbeads (diameters of 3-5 pm) interacting with platelets and reporting platelet contractile forces by fluorescence then showing ITS-coated microbeads mixed with platelet solution and added to a 96-well plate.

[0014] FIG. 3 is a graph showing fluorescence signals for PRP, PPP and PRP (Tirofiban).

[0015] FIG. 4 is a graph showing that force-sensing microspheres report aggregation force in platelet thrombi treated with aspirin (acetylsalicylic acid, ASA), a common anti-platelet drug. The result demonstrates that ITS-coated microspheres successfully respond to anti-platelet treatment.

DETAILED DESCRIPTION OF THE INVENTION

[0022] The details of one or more embodiments of the disclosed subject matter are set forth in this document. Modifications to embodiments described in this document, and other embodiments, will be evident to those of ordinary skill in the art after a study of the information provided herein. [0023] The present disclosure may be understood more readily by reference to the following detailed description of the embodiments taken in connection with the accompanying drawing figures, which form a part of this disclosure. It is to be understood that this application is not limited to the specific devices, methods, conditions or parameters described and/or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting. Also, in some embodiments, as used in the specification and including the appended claims, the singular forms “a,” “an,” and “the” include the plural, and reference to a particular numerical value includes at least that particular value, unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” or “approximately” one particular value and/or to “about” or “approximately” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment.

[0024] As used herein, the term “about,” when referring to a value or to an amount of mass, weight, time, volume, pH, size, concentration or percentage is meant to encompass variations of in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from the specified amount, as such variations are appropriate to perform the disclosed method.

[0025] It should be understood that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0026] The present invention involves force-sensing microbeads (aka “microspheres”) and a method for assessing platelet health conditions using these microbeads. These micron-sized beads can be made of various materials, including polystyrene, and are functionalized with molecular force sensors. The beads are mixed with patient platelet samples (such as platelet-rich plasma or platelet solution). Healthy platelets will aggregate with the beads, exerting contractile force that induces fluorescence in the beads. The fluorescence signals are then imaged using a microscope or quantified with a plate reader. These fluorescence signals are adopted as indicators of platelet health conditions.

[0027] Contemporary platelet function tests often lack sensitivity and specificity in detecting platelet dysfunction and related issues. The force-sensing microbeads and microbead-based platelet aggregation force assays of the present invention present a significant improvement to this technology. The assay is easy to implement, requires as little as 0.2 mL of platelet sample, and offers high sensitivity and specificity. It has been demonstrated to effectively measure platelet aggregation force and detect platelet dysfunction through force-induced fluorescence signals.

Platelet force

[0028] To achieve high sensitivity and specificity, the method described in this invention quantifies platelet force as the key indicator for evaluating platelet health. Force plays an important role in virtually all aspects of platelet functions. Following an injury, platelets initially adhere to the exposed sub-endothelium through integrins, which transmit force to mediate stable platelet adhesion and activation. The activated platelets recruit additional platelets to form thrombi and clots. Platelets further contract in the thrombi and clots to stabilize the structures, and eventually stop bleeding. During the whole process, the force transmitted by integrins (integrin 0.2(31 and integrin allbpS) play critical roles in platelet adhesion, activation, aggregation and contraction. Therefore, integrin-transmitted force is essential in normal platelet functions. The force-sensing microbeads described in this invention report platelet aggregation force by fluorescence, indicating platelet health condition with high sensitivity and specificity. In one embodiment, the microbeadbased platelet assay only requires a 0.2 mL platelet sample, which is substantially less than that required by conventional platelet function tests.

[0029] The force-sensing microbeads of the present invention enable platelet aggregation force examination (platelet force assay). Platelet adhesive forces can be detected and imaged on planar glass surfaces using a tension sensor (named integrative tension sensor, or ITS), as shown in FIGs 1 A and IB. ITS emits fluorescence if a molecular force generated by platelets ruptures the doublestranded DNA structure of the ITS and frees a fluorophore on the ITS from quenching, hence converting force signal to fluorescence signal. The structure and working principle of ITS is shown in FIG. 1A. The procedure of platelet adhesive imaging is shown in FIG. IB. FIG. 1C shows typical platelet force images after platelet plating on ITS surfaces for 1-10 min.

Integrative Tension Sensor

[0030] An Integrative Tension Sensor (ITS) directly converts molecular tensions to fluorescent signals (force-to-fluorescence conversion). As a molecular linker, the ITS is initially non- fluorescent and can be permanently activated to fluoresce by tension. The tension threshold required for ITS activation is tunable in the range of 10-60 piconewton (pN), therefore enabling the selective visualization of molecular tensions at different force levels. On a surface where integrin ligands are tethered by the ITS, integrin tensions in platelets activate the ITS and can be directly mapped by fluorescence imaging without the need of post modeling and computation which is usually required by force-to-strain approaches.

[0031] ITS is a double stranded DNA (dsDNA) with 18 base pairs. The sequences and modifications of embodiments of the single-stranded DNAs for ITS synthesis are shown below. ThioMC6-D/ denotes the thiol conjugation to the 5’ end of DNA. /BiosG/ and /Bio/ denote the DNA modifications with biotin conjugations at 5’ end and 3’ end, respectively. BHQ2 is the black hole quencher. The DNA sequences were selected and analyzed by a DNA analysis tool available from the company Integrated DNA Technologies (IDTDNA) to minimize the probability of selfdimer and hairpin formation in the ssDNA.

[0032] 5’- /ThioMC6-D/SEQ. ID 1/BHQ2/ -3’

[0033] SEQ. ID 1 : GGG CGG CGA CCT CAG CAT

[0034] 5’ - /BiosG/T/iCy3/SEQ. ID 2/ -3’

[0035] SEQ. ID 2: ATG CTG AGG TCG CCG CCC

[0036] 5’- /Cy3/SEQ. ID 2/Bio/ -3’

[0037] 5’- /Alexa647/SEQ. ID 2/Bio/ -3’

[0038] Cyclic peptide RGD with an amine group (PCI-3696-PI, Peptides International) was conjugated with the DNA strand with thiol modification. The Cyclic RGD has a PEG linker which enhance the accessibility of the RGD to integrins on cell membrane. Conjugation was conducted according to a previously published protocol. Briefly, the thiol modification on 5’ end of DNA was deprotected by TCEP (Tris(2-carboxyethyl)phosphine hydrochloride) and reacted with the maleimide group of a heterolinker SMCC-sulfo (22622, thermos scientific). The other end of SMCC-sulfo is an NHS ester (N-hydroxysuccinimide esters) which reacts with the amine of RGD. The RGD conjugated DNA was purified by electrophoresis. The 12 pN and 54 pN ITSs used in this paper were assembled by hybridizing the two ssDNAs with a concentration ratio of 1.1 : 1 (The strand with quencher: the DNA strand with fluorophore).

[0039] ITS with 54 pN threshold (with Cy3-BHQ2 pair)

[0040] 5’- /RGD/SEQ. ID 1/BHQ2/ -3’

[0041] 5’- /BiosG/T/iCy3/SEQ. ID 2/ -3’

[0042] ITS with 12 pN threshold (with Cy3-BHQ2 pair)

[0043] 5’- /RGD/SEQ. ID 1/BHQ2/ -3’

[0044] 5’- /Cy3/SEQ. ID 2/Bio/ -3’

[0045] ITS with 12 pN threshold (with Alexa647-BHQ2 pair)

[0046] 5’- /RGD/SEQ. ID 1/BHQ2/ -3’

[0047] 5’- / Alexa647/SEQ. ID 2/Bio/ -3’

Preparation of the force-sensing microbeads

[0048] As illustrated in FIG. 1A, one embodiment of the present invention shows the ITS being coated on the microbeads at a density of 100,000 ITS per bead. The ITS construct consists of 18 base-paired double-stranded DNA decorated with a biotin, a fluorophore (e.g., Atto 647N), a quencher (e.g., Black Hole quencher), and an integrin ligand (e.g., RGD peptide). Coating the beads with ITS is achieved by mixing the bead solution with the ITS solution, with the biotinstreptavidin interaction immobilizing the ITS on the bead surface.

[0049] The method of the present invention quantifies platelet contractile force using ITS-coated microbeads. In one embodiment, the microbeads have a diameter of 3-5 pm, a size similar to that of a platelet. These microbeads are either polystyrene beads (rigid) or polyacrylamide beads (soft), or beads made of other materials. The microbeads are coated with ITS through streptavidinbiotin interaction. By estimation, one microbead will be grafted with 10,000-100,000 ITS molecules. The force-sensing microbeads can be prepared on a mass scale. The microbead solution serves as a pre-prepared reagent for the platelet force assay.

Embedding force-sensing microbeads in platelet clots to report platelet functions

[0050] The ITS-coated microbeads described above are mixed up with platelet-rich plasma (or platelets resuspended in cell culture medium) and platelet agonists, such as ADP (Adenosine diphosphate). The ratio of microbead number and platelet number is in the range of 1 : 10 to 1 : 1000, dependent on desired final fluorescence signal intensity. The microbeads and platelets will form platelet clots with microbeads embedded inside. Platelets will adhere to the microbeads and the clots undergo contraction which generates contractile forces on the microbeads, eventually activating the ITS on the microbeads and making the microbeads fluorescent. The fluorescent microbeads can be either imaged by confocal microscopy to examine the contractile force distribution in the three-dimensional (3D) space, or simply read in a 96-well plate by a plate reader to report the overall platelet aggregation force signals. The large contact surface area offered by microbeads embedded in blood clots leads to high fluorescence intensity of platelet aggregation force signals, enabling the reading of such force signals directly by a plate reader.

[0051] Referring to FIG. 2, section A of the schematic shows that polystyrene microbeads (or other materials such as acrylamide) are coated with an integrative tension sensor (ITS). In one embodiment, each microbead is grafted with approximately 100,000 ITS molecules. The ITS binds to integrins on the platelet membrane, which in turn transmit platelet force to ITS and activate it to fluorescent state. The ITS construct is a double-stranded DNA decorated with an integrin peptide ligand and a fluorophore-quencher pair (e.g., Atto 647N and black hole quencher). The ITS is immobilized on the bead via covalent conjugation or biotin-avidin interaction.

[0052] Section B of the schematic shows the ITS-coated microbeads mixing with platelets at an approximate 1 : 10 ratio. The mixture is added to a plate well or a Petri-dish. Microbeads and platelets spontaneously aggregate together. Section C of the schematic shows that aggregating platelets transmit force to the ITS on the microbeads, activating the ITS to a fluorescent state by separating the DNA and removing the quencher. Section D of the schematic shows the fluorescence intensity of the microbeads being imaged using a microscope or quantified using a plate reader. The fluorescence signal is adopted to assess platelet health conditions.

[0053] In summary, the procedure of preparing this platelet force assay can be as simple as mixing microbead solution with platelet samples. After 20 min at room temperature, the sample is imaged by a microscope or quantified by plate reader, therefore having low requirement for devices or lab environment.

Utility

[0054] As platelet aggregation is a pivotal stage in hemostasis, the platelet aggregation force, reported by 3D fluorescence images or total fluorescence signal intensity of the microbeads embedded in platelet clots, serves as a biomechanical benchmark for the evaluation of platelet normal functions. This method can find application in various clinical scenarios. Examples of clinical scenarios include assessing the quality of platelets from a blood donor; valuating the quality of platelet products stored for extended periods; analyzing a patient's blood clotting capabilities to determine the necessity of platelet infusion; and the initial analysis of platelet functions of patients with suspected bleeding disorders.

There are a number of advantages of the method of the present invention. First, this method only requires a very small amount of blood, in the range of 10 pL to 100 pL. A blood sample can be provided by fingertip pricking, which is a less traumatic procedure in comparison to venous blood draw. Second, this method is also rapid, with a total assay time of 5-10 min, as platelet adhesive and contractile force signals are already significant within 5 min after platelet plating on a planar surface. Third, the microbeads embedded in platelet clots offer much greater contact surface area to platelets compared to a 2D planar glass surface, therefore generating strong fluorescence signals that can be directly read by a platelet reader without a post-signal amplification. EXAMPLES

Example 1 - Preparing an assay

[0055] An assay was prepared by mixing 20 pL of force-sensing microbeads (0.5% v/v concentration) in phosphate-buffered saline (PBS) with 180 pL of platelet-rich plasma (PRP). The mixture was then added to a 13 mm-diameter well in a glass-bottom petri dish or a well in a 96- well plate. After reacting at room temperature for 20 minutes, platelets aggregate with the forcesensing microbeads. Healthy platelets also generate force on the beads and activate ITS on the beads to a fluorescent state. The fluorescence activation is irreversible and permanently records platelet aggregation force. The sample is either imaged by a microscope or analyzed using a plate reader. The fluorescence intensity is recorded and used as a signal to indicate platelet health condition. The normal range of fluorescence intensity is pre-determined using a group of healthy platelet samples.

Example 2 - Validating the ability of force-sensing microbeads in reporting platelet aggregation force

[0056] The force-sensing microbeads have been shown to effectively report platelet aggregation force. In a typical assay, the beads were mixed with platelet -rich plasma (PRP) and added to a Petri dish. The microbeads clustered within platelet aggregates and became fluorescent. In contrast, microbeads in PRP treated with tirofiban, a potent platelet force inhibitor, did not produce fluorescence signals. Similarly, microbeads in platelet-poor plasma (PPP, in which platelets are removed) also failed to generate fluorescence signals. Fluorescence signals from microbeads in healthy platelet samples are significantly stronger than those in PPP (with platelets removed) or in PRP with platelet function inhibited by tirofiban. These results (shown in FIG. 3) demonstrate that the force-sensing microbeads can reliably report platelet aggregation force within platelet aggregates (thrombi).

Example 3 - Validating the performance of force-sensing beads in reporting platelet health conditions

[0057] The performance of platelet force assay described in this application was validated by platelets treated with acetylsalicylic acid (ASA, also known as aspirin which has been commonly adopted as an antiplatelet drug to reduce thrombosis tendency). A platelet force assay was validated using PRP treated with acetylsalicylic acid (ASA, also known as aspirin). As a positive control, force-sensing microbeads were mixed with platelet-rich plasma (PRP). A substantial number of microbeads were spontaneously incorporated into the platelet aggregates, where the microbeads become fluorescent, which can be observed as a red color. The fibrin meshwork, which assists in blood clotting, is shown as a green color. A second test was conducted using PRP treated with 100 pM aspirin for 1 hour before the force assay. The microbeads were also incorporated into the platelet clot. A third test used PRP treated with 200 pM aspirin for 1 hour before the force assay. In this case, the platelets did not appear to aggregate.

[0058] The fluorescence intensity of individual microbeads under the three different conditions was recorded. The results (shown in FIG. 4) show that microbeads in platelet samples treated with aspirin have much lower fluorescence intensities than the control. These results clearly demonstrated the feasibility of force-sensing microbeads in reporting platelet health conditions.

[0059] All documents cited are incorporated herein by reference; the citation of any document is not to be construed as an admission that it is prior art with respect to the present invention.

[0060] It is to be further understood that where descriptions of various embodiments use the term “comprising,” and / or “including” those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language "consisting essentially of’ or "consisting of.”

[0061] While particular embodiments of the present invention have been illustrated and described, it would be obvious to one skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

SEQUENCES

[0062] SEQ. ID 1 :

GGG CGG CGA CCT CAG CAT

[0063] SEQ. ID 2:

ATG CTG AGG TCG CCG CCC

Claims

What is claimed is:

1. A method of determining platelet aggregation forces comprising embedding tension sensor-coated microbeads in platelet clots and observing fluorescent signals from the microbeads that correspond to contractile forces exerted on the tension sensor-coated microbeads to report platelet aggregation forces.

2. The method of claim 1 wherein the platelet aggregation forces are used as biomechanical markers to evaluate platelet functions.

3. The method of claim 1 wherein the fluorescent signals can be read directly by a plate reader.

4. The method of claim 1 wherein platelet aggregation forces are determined using about 10 pL or less of a subject’s blood.

5. The method of claim 1 wherein platelet aggregation forces are determined using 5-10 minutes of assay time.

6. The method of claim 1 wherein the tension sensor is an Integrative Tension Sensor (ITS).

7. The method of claim 1 wherein the microbeads comprise a material selected from the group consisting of polystyrene and polyacrylamide.

8. The method of claim 1 wherein the microbeads have a diameter of 3-5 pm.

9. The method of claim 1 wherein the tension sensors are grafted to the microbeads through streptavidin-biotin interaction.

10. The method of claim 1 wherein the microbeads are used at a ratio of microbeads to platelets in the range of 1 : 10 to 1 : 1000.

11. A tension sensor-coated microbead comprising a sphere of polystyrene having a sphere surface, wherein tension sensors are grafted to the sphere surface.

12. The tension sensor of claim 11 wherein the tension sensor is an Integrative Tension Sensor (ITS)

13. The tension sensor of claim 12 wherein each microbead is grafted with from 10,000 to 100,000 ITS molecules.

14. A tension sensor-coated microbead comprising a sphere of polyacrylamide having a sphere surface, wherein tension sensors are grafted to the sphere surface.

15. The tension sensor of claim 14 wherein the tension sensor is an Integrative Tension Sensor (ITS).

16. The tension sensor of claim 15 wherein each microbead is grafted with from 10,000 to 100,000 ITS molecules.