WO2016205144A1

WO2016205144A1 - Compositions of platelet-derived theranostics and uses thereof

Info

Publication number: WO2016205144A1
Application number: PCT/US2016/037286
Authority: WO
Inventors: Gerard MARRIOTT
Original assignee: Bluecircle Therapeutics, Inc.
Priority date: 2015-06-14
Filing date: 2016-06-13
Publication date: 2016-12-22

Abstract

Aspects of the present disclosure include theranostic platelet and nano-platelet compositions comprised of blood platelets isolated from the blood of a patient or donor as (a), vehicles for molecular imaging of diseased cells, (b ), vehicles for drug-mediated therapy of diseased cells and for image and ( c ), theranostic agents for imaging and drug therapy of diseased cells. In one aspect, there is described a platelet composition comprising (i) an inactive platelet and (ii) a cargo, wherein the cargo is encapsulated within or attached to the surface of the inactivated platelet. In another aspect, there is provided a platelet composition comprising (i) a platelet, (ii) a targeting group on the surface of the platelet, wherein the targeting group specifically binds to a target, and (iii) a cargo, wherein the cargo is encapsulated within or attached to the surface of the platelet. In another aspect there is provided a platelet composition comprising (i) a nano-platelet and (ii) a cargo, wherein the cargo is encapsulated within or attached to the surface of the nano-platelet.

Description

COMPOSITIONS OF PLATELET-DERIVED THERANOSTICS AND USES

THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority benefit of United States Provisional Patent application No. 62/175,346, filed June 14, 2015; of United States Provisional Patent Application No.

62/220,200, filed September 17, 2015; and of United States Provisional Patent Application No. 62/311,743, filed March 22, 2016. The entire contents of each of the applications are hereby incorporated by reference herein.

BACKGROUND

[0002] Systems that provide for simple and effective targeting and delivery of cytotoxic drugs to diseased tissue including tumor cells, and high-contrast optical or magnetic signals of the same targeted cells in humans, are highly valued for quantitative detection, diagnosis and treatment of cancer and other diseases.

[0003] In the case of cancer for example, there is a pressing need to develop detection systems that can reveal the location of tumor cells at primary and secondary growth sites, and metastatic cells with a signal to noise that reveals the loci of individual migrating tumor cells. Moreover, it would be desirable to integrate within these detection systems the capability to deliver a cargo of cytotoxic drugs to targeted cells. While many systems have been described to target and to image and/or deliver cytotoxic drugs to targeted cells, these systems are limited by factors including: a), short circulation times in the blood that prevent them from binding in significant numbers to the target cells, which is usually compensated for by injecting very high dosages of the vehicle, likely causing liver or other organ damage; b), off-target effects associated with high dosage of the cytotoxic cargo, which may overload the capacity of the body to accommodate and to inactivate their cytotoxins; c), they are typically excluded from entering brain tissue because they do not cross the blood brain barrier (BBB); d), they may accumulate through aggregation within off-target sites with unwanted leakage of cytoxins and tissue damage. BRIEF SUMMARY OF THE INVENTION

[0004] Provided herein are platelet compositions comprising an inactive platelet and a cargo, wherein the cargo is encapsulated within or attached to the surface of the inactivated platelet.

[0005] Further provided are platelet compositions comprising a platelet, a targeting group on the surface of the platelet, wherein the targeting group specifically binds to a target, and a cargo, wherein the cargo is encapsulated within or attached to the surface of the platelet. In some embodiments, the platelet is an inactive platelet.

[0006] Also provided herein are platelet compositions comprising a nano-platelet and a cargo, wherein the cargo is encapsulated within or attached to the surface of the nano-platelet. In some embodiments, the nano-platelet has a diameter of about 1000 nm or less. In some embodiments, the nano-platelet has a diameter between about 30 nm and about 500 nm. In some embodiments, the nano-platelet has a diameter between about 150 nm and about 250 nm. In some

embodiments, the nano-platelet is derived from an activated platelet. In some embodiments, the nano-platelet is derived from an inactive platelet. In some embodiments, the nano-platelet is formed by sonicating, extruding, or enzymatic treatment of the platelet.

[0007] In some embodiments of the platelet composition described herein, the platelet composition further comprises a targeting group on its surface, wherein the targeting group specifically binds to a target. In some embodiments, the target is a target cell, a target structure, or a soluble molecule. In some embodiments, the target is a cancer cell, such as a multiple myeloma or a leukemia. In some embodiments, the target is an atherosclerotic plaque or an amyloid plaque. In some embodiments, the targeting group is transferrin, a polypeptide ligand, a small molecule ligand, an antibody, or an engineered scaffold that mimics an antibody. In some embodiments, the targeting group is a polypeptide. In some embodiments, the polypeptide is expressed from a recombinant nucleic acid introduced into the platelet.

[0008] In some embodiments of the platelet composition described herein, the cargo comprises a therapeutic agent or a diagnostic agent. In some embodiments, the cargo comprises a therapeutic agent and a diagnostic agent. In some embodiments, the cargo comprises a magnetic nanoparticle. In some embodiments, the cargo comprises a cytotoxic drug. In some embodiments, the cargo comprises a fluorescent probe, a photodynamic therapy probe, a nanoparticle, a polymer, or an MRI contrast agent. In some embodiments, the cargo comprises recombinant nucleic acid. In some embodiments, the recombinant nucleic acid encodes a therapeutic agent or a diagnostic agent. In some embodiments, the recombinant nucleic acid is encapsulated within the platelet or the fragment of the platelet. In some embodiments, the recombinant nucleic acid is coupled to the surface of the platelet or the fragment of the platelet. In some embodiments, the recombinant nucleic acid is recombinant mRNA or recombinant cDNA.

[0009] In some embodiments of the platelet composition described herein, the platelet is inactivated by contacting the platelet with an inactivating compound. In some embodiments, the inactivating compound is kabiramide C, aspirin (or salicylic acid), or prostaglandin E2. In some embodiments, the inactivating compound is cytochalasin D.

[0010] Further provided is a composition comprising a polymer and the platelet composition described herein. In some embodiments, the polymer is a hydrogel. In some embodiments, the polymer is poly(N-isopropylacrylamide).

[0011] Also described herein are devices for the manufacture of a platelet composition, the device comprising a chamber; a microfluidic conduit; one or more reagent ports fluidly connected to the microfluidic conduit; and a concentrator, wherein the microfluidic conduit connects the chamber and the concentrator. In some embodiments, the device further comprises a sonicator. In some embodiments, one or more reagent ports provides an inactivating compound, a targeting group, a diagnostic agent, and/or a therapeutic agent. In some embodiments, the chamber is a culture chamber. In some embodiments, the culture chamber comprises a matrix that supports cellular growth. In some embodiments, the matrix that supports cellular growth comprises a hydrogel.

[0012] Further described herein are methods of forming the platelet composition described herein comprising contacting a platelet with an inactivating compound; and loading the inactive platelet with the cargo or attaching the cargo to the surface of the inactive platelet.

[0013] Also described are methods of forming the platelet composition comprising loading a platelet with the cargo or attaching the cargo to the surface of a platelet; and contacting the platelet with an inactivating compound.

[0014] Further provided are methods of forming the platelet composition comprising attaching the targeting group to a surface of a platelet; and loading the platelet with the cargo or attaching the cargo to the surface of a platelet. [0015] Also provided are methods forming the platelet composition comprising fragmenting a platelet to form a nano-platelet; and loading the nano-platelet with the cargo or attaching the cargo to the surface of a platelet.

[0016] The present application further describes methods forming the platelet composition comprising loading a platelet with the cargo or attaching the cargo to the surface of a platelet; and fragmenting the platelet to form nano-platelets.

[0017] In some embodiments of any of the methods described herein, the method further comprises culturing a cytomegakaryocyte to produce the platelet. In some embodiments, the method further comprises transfecting the cytomegakaryocte with a recombinant nucleic acid. In some embodiments, the recombinant nucleic acid is recombinant mRNA or recombinant cDNA. In some embodiments, the recombinant nucleic acid encodes a targeting group, a therapeutic agent, or a diagnostic agent.

[0018] Also provided herein are methods of treating a disease in an individual comprising administering to the individual the platelet composition described herein. Further described are methods of treating a disease in an individual comprising administering to the individual the composition comprising a polymer and the platelet composition described herein. The present application also describes methods of diagnosing a disease in an individual comprising administering to the individual the platelet composition described herein. Further described herein are methods of diagnosing and treating a disease in an individual comprising

administering to the individual the platelet composition described herein. In some embodiments, the platelet composition comprises a diagnostic agent and a therapeutic agent. In any of the methods of treating or diagnosing a disease in an individual, the disease can be cancer,

Alzheimer's disease, or atherosclerosis.

[0019] Further provided herein are methods of producing the platelet composition described herein by using the device described herein by contacting a platelet with an inactivating compound; and loading the inactive platelet with the cargo or attaching the cargo to the surface of the inactive platelet. In some embodiments, the methods further comprise culturing cytomegakaryocytes in the chamber to produce platelets. In some embodiments, the methods further comprise attaching a targeting group to the surface of the platelet, wherein the targeting group specifically binds to a target. In some embodiments, the methods further comprise fragmenting the platelets. In some embodiments, the methods further comprise sonicating the platelets. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 illustrates the "arm back to arm" approach to diagnose and treat patients with cancer, cardiovascular and neurodegenerative diseases using their own platelets as the source of theranostic platelets.

[0021] The images in Figure 2A show purified platelets loaded with TMR-KabC (DIC, fluorescence and merged images). The image shown in Figure 2B shows the optimal condition for efficient labeling of platelets as quantified by FACS analysis of TMR-KabC loaded platelets.

[0022] The effect of thrombin on platelet activation is shown in Figure 3A. The scanning electron microscope image of platelets treated with KabC show no evidence of thrombin- activation whereas platelets without KabC activate normally. In the transmission electron microscope images shown in Figure 3B, the KabC treated platelets have a normal complement of intracellular vesicles and a smooth external membrane whereas the thrombin treated platelets generate characteristic filopodial like extensions and loss of intracellular vesicles.

[0023] The size distribution of platelets and their fragmented forms are shown in Figure 4A and compared to that with 200 nm reference beads. The sonicated platelets treated with KabC have a smaller size than the non- sonicated control platelets with an average size similar to that of the 200 nm beads. In the transmission electron microscope images in Figure 4B, the decrease in average in the size of the sonicated vesicles compared to the intact platelet is apparent with the nano-platelets showing vesicles and closed membranes.

[0024] Figure 5A shows a plot of a FACS analysis of ,in intact platelets treated with KabC showing saturation of doxorubicin loading at 0.1 mg/ml. The FACS plots in Figure 5B show almost no fluorescein emission in unloaded platelets (left) and far stronger emission in platelets treated with 0.1 mM CFDA

[0025] Figure 6:A shows SDS-PAGE analysis of Cy5 labeling of transferrin. Left image of Figure 6A shows the transmission of the gel for product of a reaction mixture composed of transferrin (Sigma) with Cy5-NHS (GE Healthcare) showing dominant coloration at 80kD corresponding to the mass of transferrin. The right image of Figure 6A shows the Coummassie stain of the same gel confirming the location of the transferrin. In the absorption spectrum shown in Figure 6B, the Cy5 absorption is used to estimate a labeling ratio for Cy5 transferrin of almost 5: 1. [0026] Figure 7A shows a FACS analysis of the reaction between Cy5-transferrin co-labeled with MBS and Iminothiolane treated platelets. The labeling of platelets with Cy5-transferrin is strong and saturated under the conditions applied in this study. The images in Figure 7B show that the fluorescence of Cy5-transferrin overlaps with the location of platelets and that every platelet in the field is labeled with Cy5-transferrin.

[0027] Figure 8 shows an overlay of the bright field and Cy5 fluorescence image of Cy5- transferrin labeled platelets in a field of MM2 cancer cells. Individual platelets are seen to bind to the MM2 membrane and the presence of red (Cy5) fluorescence inside the MM2 cells indicates the theranostic platelets are endocytosed.

[0028] Figure 9A shows an overlay of the bright field and Cy7 fluorescence image of a mouse bearing a MM2 tumor that was injected with a suspension of Cy7-transferrin (short arrow) coupled theranostic platelets as a function of time. The Cy7 emission is seen throughout the mouse over the first 48 hours including the brain, which indicates the Cy7 labeled theranostic platelets can cross the blood brain barrier. The Cy7 emission is largely cleared after 7 days and is localized, as seen from Cy7 fluorescence images of the excised organs, in the tumor shown inf Figure 9B. The Cy7-fluorescence probe is cleared by the liver and spleen and also shows up in the kidneys.

[0029] Figure 10A shows an overlay of the bright field and Cy7 fluorescence image of a mouse bearing a MM2 tumor that was injected with a suspension of Cy7-transferrin (short arrow) coupled theranostic nano-platelets as a function of time. The Cy7 emission is seen throughout the mouse over the first 48 hours including the brain, which indicates the Cy7 labeled nano-platelets can cross the blood brain barrier. The Cy7 emission is largely cleared after 7 days and is localized as seen from Cy7 fluorescence images of the excised organs in the tumor shown in Figure 10B. The fluorescence stain is cleared by the liver and spleen and shows up in the kidneys and faeces.

[0030] Figure 11 A shows schematics of devices that carry out of the loading and chemical reactions necessary for the production of theranostic platelets. Figure 1 IB shows schematics of devices that carry out of the loading and chemical reactions necessary for the production of nano-platelets. The device uses purified platelets isolated from the patient as the input and carries out sequential loading with KabC, CFDA or a NIR dye, Doxorubicin, iminothiolane and coupling with the maleimide congugate of the produces the final form of the theranostic platelet or nano-platelet that is ready to inject into the same patient. [0031] Figure 12A shows schematics of devices that carry out of the production of platelets, and the loading and chemical reactions necessary for the production of theranostic platelets. Figure 12B shows schematics of devices that carry out of the production of platelets, and the loading and chemical reactions necessary for the production of nano-platelets. Human cytomegakaryocytes transfected with a NIR probe (mRuby) and a surface localized capture group that may be an engineered antibody, transferrin or another capture ligand such as

Endostar. The device allows for automatic control of the loading of KabC and the toxin such as doxorubicin carries out of the loading and chemical reactions necessary for the production of theranostic platelets and nano-platelets. The final form of the theranostic platelet or nano-platelet that is ready to inject into the same patient.

[0032] Figure 13 shows a schematic of a method to bias the distribution of theranostic platelets to the site of a tumor. Theranostic platelets are mixed with the sol state of a hydrogel (NIP AM) and injected in liquid form to the site believed to harbor a tumor or damaged tissue. The higher body temperature results in a stiffening of the hydrogel and entrapment of theranostic platelets that leach from the polymer slowly at the site of the tumor.

[0033] Figure 14 shows the results of studies related to the production and characterization of theranostic platelets. In sub-figure (a), TMR-fluorescence image showing accumulation of TMR-KabC in human platelets, in sub-figure (b), SEM image of KabC -platelets exposed to thrombin; in sub-figure (c), SEM image of human platelets without KabC exposed to 1 U/ml thrombin; in sub-figure (d), Image of doxorubicin fluorescence in KabC-platelets; in sub-figure (e), Overlay image of phase-contrast and Cy5-transferrin fluorescence of KabC-platelets in sub- figure (f), FACS analysis of 30μΜ chlorin-E6 in KabC-platelets recorded 24-hours after loading chlorin-E6; Tl MRI- images of in sub-figure (g), Control KabC-platelets and in sub-figure (h), KabC-platelets passively loaded with Magnevist in a sample well; in sub-figure (i), a photograph of KabC-platelets (endocytosed magnetic nanoparticles) that are magnetically-trapped against the wall of the tube; T2 MRI-images of in sub-figure (j), Control KabC-platelets and in sub- figure (k), KabC-platelets endocytosed with magnetic nanoparticles in a sample well (1) A single frame from a movie of overlapped fluorescence images of DAPI in RPMI cells and Cy5- transferrin on KabC-platelets recorded 45-minutes after adding 10 platelets/ml. Cy5-KabC- platelets bind occasionally to the surface of RPMI cells but are rarely seen to be endocytosed. The Cy5-fluorescence in the cytoplasm is weak but evident in the cytoplasm. [0034] Figure 15 shows the preparation and characterization of theranostic nano-platelets. In sub-figure (a), SEM image of a single sonicated platelet with a closed membranes and numerous surface projections; In sub-figure (b), TEM image of sonicated KabC-platelets showing a mother platelet (M), and daughter (D) platelets with similar intracellular staining and irregular surfaces. FACS analysis of light- scattering for intact platelets in sub-figure (c); in sub-figure (d), nano- platelets generated from sonication, and in sub-figure (e), size-distribution of 200nm beads. FACS analysis of doxorubicin-fluorescence in RPMI cells that have endocytosed transferrin- linked nano-platelets; in sub-figure (f), nano-platelets without doxorubicin; in sub-figure (g), nano-platelets previously incubated with doxorubicin at 2mg/ml but without transferrin; in sub- figure (h), nano-platelets with transferrin previously incubated with doxorubicin at 2mg/ml with surface-linked transferrin. The mean intensity of doxorubicin fluorescence in RPMI cells increases 2.54-fold between transferrin-linked nano-platelets and nano-platelets without transferrin; in sub-figure (i), a single frame from a movie of overlapped fluorescence images of DAPI in RPMI cells and Cy5-transferrin on nano-platelets recorded 45-minutes after adding 10 platelets/ml.

[0035] Figure 16 shows the performance of theranostic platelets in targeting multiple myeloma in immuno-compromised mice. Sub-figure (a) shows images of a time course of Cy7- fluorescence in a mouse bearing a sub-cutaneous RPMI cell tumor on its back and injected with Cy7/transferrin platelets on day- 15. RBMI cells injected intra-cranially into mice on day 1 are injected with Cy7/transferrin platelets on day-5 and Cy7-fluorescence recording on day-6. Cy7- fluorescence images recorded in sub-figure (b), a control mouse ie no tumor; in sub-figure (c) and sub-figure (d), mice injected intra-cranially with RPMI cells. The mouse shown in sub- figure 3c was imaged by MRI on day-8; in sub-figure (e) Tl image recorded before and, in sub- figure (f), after injecting Magnevist in the tail vein. The look up tables presented has units of radiant efficiency (photons/sec/cm 2 /steradian)/^W/cm 2 ).

[0036] Figure 17 shows performance of theranostic nano-platelets in targeting multiple myeloma in immuno-compromised mice. Sub-figure (a) shows images of the time course of Cy7-fluorescence in a mouse bearing a sub-cutaneous RPMI cell tumor on its back and injected with Cy7/transferrin nano-platelets on day- 15. Cy7-fluorescence imaging of Cy7/transferrin nano-platelets in a mouse recorded 9-days after intra-cranial injection of RPMI cells. Cy7- fluorescence images recorded in sub-figure (b), a control mouse ie no tumor; in sub-figure c), Cy7-fluorescence and bright field overlap image of tumor mouse after exposing the skull and underlying brain; in sub-figure d), Cy7-fluorescence image of the excised brain spliced into the left and right sides and showing fluorescence is restricted to the right side; in sub-figure (e), photograph of the sliced brain indicating the site of RPMI cell injection.

[0037] Figure 18 shows in sub-figure (A), MRI-images of PBS and KabC-platelets with and without loaded Magnevist with the corresponding Tl values for each sample; shows in sub- figure (B), MRI-images of PBS and KabC-platelets with and without endocytosed magnetic particles with the corresponding T2 values for each sample

[0038] Figure 19 shows in the sub-figures, FACS analysis of the optimized sonication to prepare Nano-platelets.

[0039] Figure 20 shows in (a), Analysis of Cy7/transferrin nano-platelets as targeting probes for RPMI cell-derived multiple myeloma in mice. Cy7-fluorescence imaging of theranostic nano-platelets in mice 13-14 days after intra-cranial injection of RPMI cells: Cy7-fluorescence in a mouse with multiple myeloma in the cranial cavity recorded after (a) 28-hours and 48-hours after injecting theranostic nano-platelets. (b), Cy7-fluorescence of theranostic nano-platelets in a control mouse (no cranial multiple myeloma) after 28-hours and 48-hours; (c), An overlay image of bright- field and Cy7-fluorescence of a control mouse (no tumour) and (d) the mouse with intra-cranial multiple myeloma after removing skin and tissue above their skulls, (e), Bright- field photograph of the exposed brain tissue of the mouse with multiple myeloma, and (f) a photograph of the separated brain and multiple myeloma.

[0040] Figure 21 shows MRI images of the mouse from figure 3d before (A), and after (B) injecting Magnevist into the tail vein. (C), Cy5.5-fluorescence image of Cy7/transferrin linked KabC-platelets loaded with chlorin-E6 that were injected into a control mouse (no tumour) and a mouse 5-days earlier with RPMI cells. The fluorescence originates from chlorin E6 (not Cy7). The background fluorescence in the mouse is somewhat higher at the shorter wavelength used to excite chlorin-E6.

[0041] Figure 22 shows an alternate mode to bring about tumor cell death employs human platelets as a platform for antibody based approaches to immuno-therapy. Platelets covalently linked with components of dual-affinity retargeting therapy (DART) or tetravalent tandem diabodies (TandAbs). In the DART mimetic, platelets are labeled with a specific antibody (or transferrin) for targeting to tumor cells. The same platelets are chemically linked to a T-cell recruiting and activating protein. In TandAbs platelets are linked with the targeting protein and upto 3 proteins that target activated T-cells, or else with combinations of targeting protein and activated T-cells targeting/activation proteins. The figure depicts different populations of platelets harboring the targeting protein and a single activated T-cell recruitment and activation protein, or the targeting protein and two activated T-cell recruitment and activation proteins or an additional targeting protein and a single activated T-cell recruitment and activation protein. The DART or TandAbs coupled platelets act as bridges that mediate activated T-cell-mediated targeting and killing of tumor cells. The presence of multiple copies of the targeting protein will increase binding affinity through avidity, while multiple T-cell recruiting and activating proteins linked on the same or other platelets harboring the targeting protein will help to augment the recruitment and activation of T-cells on the tumor cell.

[0042] Figures 23A, 23B, 23C, 23D, 23E, and 23F show the characterization and analysis of targeted tumor-targeting platelets. Figure 23A shows confocal fluorescence image showing the accumulation of TMR-KabC in a field of human platelets. Bar = 10 μιη. Figure 23B shows FACS analysis of TMR-KabC loading of purified human platelets recorded 24-hours after incubating with 10 platelets/ml with TMR-KabC at 5 μΜ. Figure 23C shows confocal fluorescence image of the intrinsic fluorescence of EPI in KabC-platelets. Bar = 10 μιη. Figure 23D shows confocal fluorescence image of KabC-platelets loaded with CFDA. CFDA is de- esterified within the platelet to produce a fluorescent di-anionic fluorescein probe that is trapped in the cytosol. Measure bar = 10 μιη. 23E. FACS analysis of KabC-platelets without any probe labeling. 23F. FACS analysis of KabC-platelets loaded with a 30μΜ solution of chlorin-e6 in KabC-platelets and recorded 24-hours later.

[0043] Figures 24A, 24B, 24C, 24D, 24E, and 24F show the characterization of KabC- stabilized platelets. Figure 24A shows SEM image of a KabC-platelets previously exposed to thrombin (lU/ml). Figure 24B shows TEM image of a single KabC-platelet previously exposed to thrombin (lU/ml). Figure 24C shows SEM image of human platelets without KabC previously exposed to 1 U/ml thrombin. 24D. TEM image of a single human platelet without KabC previously exposed to 1 U/ml thrombin. FACS analysis of light- scattering for KabC- platelet loaded with EPI 24E. before, and 24F. after an exposure to lU/ml thrombin.

[0044] Figure 25: Characterization of transferrin conjugates and their coupling to KabC- platelets. SDS-PAGE images of transferrin labeled with Cy5 and PDM showing 25A. the unstained gel with the light blue color originating from absorption of transferrin linked Cy5- probes, and 25B. the same gel stained with Coumassie Blue. Cy5-labeled transferrin shows up as band at ~80kD in both cases. 25C. Absorption spectrum of the transferrin of Cy5/PDM. Analysis of the absorption data at 650nm (Cy5) and 280 nm (transferrin) is used to calculate a labeling ratio of 4.5 Cy5 molecules per transferrin molecule. 25D. Overlay of the phase-contrast and Cy5-fluorescence images of KabC-platelets coupled with transferrin conjugated to Cy5/PDM. Bar = 10 μιη 25E. FACS analysis of the background fluorescence of KabC-platelets measured in the Cy5-emission channel. 25F. FACS analysis of the optimized loading condition developed to couple transferrin conjugated to Cy5/PDM to thiol-containing KabC-platelets measured in the Cy5-emission channel.

[0045] Figure 26: Characterization of interactions between KabC-platelets and tumor cells. 26A. FACS analysis of RPMI8226 cells stained directly with a FITC-conjugated antibody against human transferrin receptor. 26B. FACS analysis of K562 cells stained directly with a FITC-conjugated antibody against human transferrin receptor. 26C. Confocal Cy5-fluorescence image of surface-attached RPMI 8226 cells recorded 8-hours after being incubated with Cy5- coupled KabC-platelets. Bar = 10 μιη 26D. Confocal Cy5-fluorescence image of surface- attached RPMI 8226 cells recorded 8-hours after being incubated with KabC-platelets coupled with Cy5 and transferrin. Bar = 10 μιη 26E. Higher-resolution confocal Cy5-fluorescence image of surface- attached RPMI8226 cells recorded 8-hours after being incubated with KabC-platelets coupled with Cy5 and transferrin showing individual surface attached platelets and clusters of Cy5-fluorescence (cyan arrows). Bar = 5 μιη 26F. Confocal Cy5-fluorescence image of surface- attached K562 cells recorded 8-hours after being incubated with Cy5-coupled KabC-platelets. Bar = 10 μιη 26G. Confocal Cy5 -fluorescence image of surface-attached K562 cells recorded 8- hours after being incubated with KabC-platelets coupled with Cy5 and transferrin. Bar = 10 μιη 26H. Higher-resolution confocal Cy5-fluorescence image of surface-attached K562 cells recorded 8-hours after being incubated with KabC-platelets coupled with Cy5 and transferrin showing individual surface attached platelets and clusters of Cy5-fluorescence (cyan arrows).

[0046] Figure 27: in vivo Cy7-imaging of platelets in control and tumor-bearing mice. 27 A. Time course of Cy7-fluorescence images of a mouse bearing a sub-cutaneous RPMI8226 xenograft on its back injected with Cy7 coupled KabC-platelets on day- 15. The look up tables presented has units of radiant efficiency (photons/sec/cm 2 /steradian)/^W/cm 2 ). 27B. Time course of Cy7-fluorescence in a mouse bearing a sub-cutaneous RPMI cell xenograft on its back and injected with Cy7/transferrin coupled platelets on day- 15. 27C. Bar graph showing the integrated intensities of Cy7-fluorescence in select organs excised from mice injected with Cy7- coupled platelets with transferrin (test) or without transferrin (control).

[0047] Figure 28: in vivo Cy7-imaging of platelets in control mice and mice bearing intracranial myeloma xenograft. Cy7-fluorescence images of mice bearing intra-cranial xenografts that formed after injecting RPMI8226 cells. The mice (n=3) were injected with Cy7/transferrin coupled KabC -platelets on day-5 and the Cy7-fluorescence imaged after 24-hours. Cy7- fluorescence images from two of these mice are shown in 28A and 28B show structured fluorescence from their crania. 28C. Cy7-fluorescence image of a control mouse without xenograft injected with Cy7 /transferrin coupled KabC-platelets. The look up tables presented has units of radiant efficiency

The mouse shown in Figure 28 A was imaged by MRI on day- 8; 28D. Tl image recorded before and, 28E after injecting Magnevist in the tail vein. In a second control group, mice were injected with U87 cells transfected with RFP to allow for in vivo imaging of neuroglioblastoma xenograft. 28F. RFP fluorescence from a representative mouse in this group showing the locus of the

neuroglioblastoma xenograft (yellow arrow). 28G. corresponding Cy7-image of the same mouse injected with KabC-platelets coupled with transferrin and Cy7. Cy7 emission was not detected in the brain of this or other mice in the group. 28H. FACS analysis of U87 cells stained directly with a FITC-conjugated antibody against human transferrin receptor showing the majority of U87 cells express low levels of transferrin receptor.

[0048] Figure 29: Immuno-histochemical analysis of RPMI8226 xenograft slices. Tumor slices excised from mice injected with KabC-platelets coupled with transferrin and chlorin e6. Measurement bars in all images is 50 μιη: 29A. in vivo red-fluorescence image of a

representative mouse bearing an intra-cranial xenograft that developed after injecting RPMI8226 cells and then injected with KabC-platelets loaded with chlorin e6. 29B. in vivo red-fluorescence image of a representative mouse bearing an intra-cranial xenograft that developed after injecting RPMI8226 cells and then injected with KabC-platelets coupled with transferrin and loaded with chlorin e6. 29C. Representative confocal fluorescence image of a FITC-labeled antibody directed against human transferrin receptor showing the distribution of RPMI8226 cells in the tumor slice. 29D. Bright-field image of the same tumor slice. 29E. Red-fluorescence image of the xenograft slice showing the distribution of platelets in blood vessels and the tumor mass. 29F. overlay of the FITC, bright- field and red-fluorescence images showing infiltration of platelets within the tumor mass.

[0049] Figure 30: FACS analysis of TMR-KabC loading of human platelets. FACS analysis of TMR-KabC loading of purified human platelets 24 hours after incubating defined concentrations of TMR-KabC with 1088 platelets in 1 ml of platelet buffer.

[0050] Figure 31: FACS analysis of the chemical-coupling of PDM/Cy5-transferrin to thiol- containing KabC-platelets. FACS analysis of the optimized loading condition for Cy5/PDM transferrin coupling to thiol-containing KabC-platelets. The photograph on the bottom left shows an Eppendorf tube containing a suspension of KabC-platelets coupled on their surface with Cy7- NHS that had been allowed to settle by gravity. Sedimented platelets are heavily labeled with Cy7 as can be seen by the strong absorption of far red light by Cy7 molecules.

[0051] Figure 32: in vivo imaging of platelets in myeloma xenotransplants. 32A and 23B. KabC-platelets coupled with Cy7 and transferrin were injected into the tail veins of two control mice without RPMI8226 cells. 32C. KabC-platelets coupled with Cy7 and transferrin were injected into one of the three mice that 5-days earlier had been injected intra-cranially with RPMI8226 cells. 32D and 32E. KabC-platelets coupled with Cy7 and transferrin were injected into the tail veins of two mice that had been injected intra-cranially with RFP-transfected U87 cells. 32F and 32G. Chlorin e6-loaded KabC-platelets lacked transferrin were injected into the tail veins of two mice that had been injected intra-cranially with RPMI8226 cells. 32H and 321. Chlorin e6-loaded KabC-platelets surface-coupled with transferrin were injected into the tail veins of two mice that had been injected intra-cranially with RPMI8226 cells.

DETAILED DESCRIPTION

[0052] Aspects of the present disclosure include theranostic platelet and nano-platelet compositions comprised of blood platelets isolated from the blood of a patient or donor as (a), vehicles for molecular imaging of diseased cells, (b), vehicles for drug-mediated therapy of diseased cells and for image and (c), theranostic agents for imaging and drug therapy of diseased cells. The compositions and methods to prepare theranostic platelets and their nano-platelet derived particles within an hour of drawing blood include a cytosolic actin binding drug or aspirin or prostaglandin to inhibit platelet activation, a cytosolic-localized cytotoxic drug to kill the target cell, cytosolic or surface- attached detection probes or nanoparticles to image the distributions of theranostic platelets and nano-platelets in the body by NIR-fluorescence, Tl and T2 MRI signals, surface plasmon resonance and ultrasound. Also provided are methods to link capture ligands and proteins and therapeutic antibodies that bind to target protein on diseased cells to the platelet surface. Also provided are methods to express targeting proteins and detection proteins for NIR-fluorescence and MRI from mRNA or cDNA transfected within the platelet cytosol. Also provided are theranostic platelets linked at their surface with proteins that induce endocytosis on binding to the target cell. Also provided are entrapped- or surface-linked magnetic-nanoparticles that direct theranostic platelets and nano-platelets via an external magnetic field to target tissue wherein the theranostic platelets are imaged by MRI, and wherein at higher magnetic fields the cytotoxic cargo of the target-bound theranostic platelets or nano- platelets are released by magnetic heating of the nanoparticles. Also provided are theranostic platelets and nano-platelets that harbor detection probes including small molecule and nanoparticle probes including nano-bubbles for in vivo imaging including signal detection from changes in the NIR-fluorescence, bioluminescence, magnetic field, MRI, SPR, and ultrasound signal. Also provided are methods to prepare manually and by way of a device theranostic platelets and nano-platelets from human blood and within a device that supports the growth of cytomegakaryocytes (CMK) that are engineered for the automated production of theranostic platelets and nano-platelets. Theranostic platelets and nano-platelets are designed for broad based imaging, diagnosis and treatment of diseased states whose therapy is best realized by inducing apoptosis or disruption of a protein aggregate including cancer and Alzheimer's.

[0053] Overcoming limitations of prior compositions and methods represents a significant advance in the detection, imaging and treatment of cancer and other human diseases including Alzheimer's disease (AD) and arteriosclerotic heart disease. In this filing we describe the design, preparation, characterization and applications of theranostic agents that are derived from human platelets. The theranostic platelets are employed as illustrated in figure 1 in an "arm back to arm" approach that allows one to generate human platelets or platelets -derived nano-platelets that are repurposed to function as vehicles for (a), targeted molecular delivery and imaging and detection of a disease, (b), vehicles for targeted molecular treatment of a disease, and (c), vehicles that combine targeted delivery, imaging and treatment hereafter referred to as theranostic platelets. In each of the three classes of repurposed platelets the original blood platelets are isolated from the patient to be treated and reconfigured via a series of optimized loading and labeling methods to introduce into the platelet specific molecular functionalities for targeting, imaging and/or treatment. These re-purposed platelets are then injected into the same patient to image tumor cells and other targets, while potent cytotoxins embedded within the platelet are delivered to and released within or in the vicinity of the target cell by natural endocytosis, or by optical, ultrasound or magnetic perturbations applied to the region labeled with the theranostic platelet, to bring about cell death. The advantage of using platelet-derived agents as theranostic agents over nanoparticle based agents include their ready availability from donated blood, and easiness in loading and labeling with drugs, targeting groups and detection probes using mild conditions. More importantly, since theranostic platelets are isolated from the same patient being treated, the rejection and clearance rate is significantly reduced compared to that in the case of nanoparticle based agents.

[0054] Platelets are sub-cellular fragments produced by cytomegakaryocytes that circulate in the vascular system where they play essential roles in hemostasis and inflammation. Activated platelets were found in tumors more than 150 years ago, although relatively few studies have addressed their roles in tumor microenvironments.

[0055] Platelets gain access to the tumor microenvironment by passive diffusion across leaky capillaries, and via associations with neutrophils and other immune cells. As described herein, platelets can be engineered for in vivo imaging of tumor cells, and for targeted delivery of protein or small molecule therapeutic agents to targeted cells. The methods described herein include methods to (a), inhibit platelet aggregation; (b), to link specific antibodies or protein ligands to the platelet surface for tumor- targeting; (c), to load the platelet cytosol with detection probes for in vivo imaging; and (d), to load the platelet with cytotoxins for drug delivery.

Repurposed platelets offer a number of advantages over artificial nanoparticles for in vivo targeting and imaging of tumor cells. First, platelets are recognized by the host and can be routinely transfused in cancer patients. Second, platelets have privileged access to the tumor microenvironment. Third, platelets circulate in vivo for an extended period of time (such as up to 9-days), whereas nanoparticle-derived vehicles are efficiently endocytosed by macrophages and typically circulate for only 3-5 hours, which reduces their change to interact with target cells, especially those deep in a tumor. The long circulation time of injected platelets increases the chance of encounters with targeted tumor cells. The short circulation is especially problematic for nanoparticles loaded with cytotoxins, as the sudden appearance of a high concentrations of the cytotoxin in the liver may overwhelm detoxification pathways and result in liver damage. Fourth, human platelets are much larger than nanoparticles and may accommodate more than about 100-fold more molecules of a surface-coupled targeting protein, and more than about 1000-fold more molecules of an internalized detection probe or therapeutic drug.

[0056] A major challenge in repurposing human platelets as tumor-targeting agents is the need to overcome their tendency to undergo non-specific activation and aggregation. Platelet- activation is characterized by the formation of numerous actin polymerization-driven membrane protrusions that promote platelet-aggregation and clumping. This aggregation reaction prevents platelets from being used as stand-alone vehicles for tumor- targeting. This disclosure presents elegant approaches to suppress both specific and non-specific platelet- aggregation, one of which involves loading human platelets with kabiramide C (KabC), a natural product, membrane permeable drug that binds tightly to the barbed-end of the actin filament where it effectively inhibits actin polymerization. KabC-loaded platelets do not produce membrane protrusions or aggregate on exposure to thrombin, or during manipulations used in their transformation to tumor- targeting vehicles.

[0057] As described herein, platelet-access to tumors was exploited to repurpose platelets as living vehicles for targeting and imaging tumors. In some embodiments, aggregation- incompetent human platelets, surface-coupled with transferrin bind specifically to multiple myeloma cells (such as RPMI8226 cells) and leukemia cells (such as K562 cells) that over- express transferrin receptor. The platelets modified for tumor- targeting, in vivo imaging and drug-delivery can be adapted for a variety of surface-coupled antibodies, detection probes and therapeutic agents for in vivo targeting of cells associated with cardiovascular and neurologic diseases.

[0058] A convenient method to prepare theranostic platelets and nano-platelets is to inhibit platelet activation, which is realized by either adding kabiramide C, a small molecule cell permeable drug that caps the barbed-ends of actin filaments to fresh platelets, or else by adding aspirin (or salicylic acid) to the purified platelets, or else by adding prostaglandin. This initial step as shown in figures 2, 3,14a,b) allows for high-yield production of theranostic platelets, which are produced by sequential steps that include passive loading of small cytotoxic drugs, including doxorubicin (Figures 5a, 14d) and detection probes including passively-loaded NIR- fluorescent cyanine dyes, including dihydro-Cy7 and Ros-Star 800, Chlorin E6, (Figure 15f) and fluorogenic probes whose fluorescent state is generated after de-esterification in the platelet cytosol (Figure 5b). KabC -platelets can also be loaded passively and stably with MRI contrast agents including Magnevist (Figure 14g,h; 18a,b). Also included here are reactions that chemically-link targeting agents such as transferrin, antibodies against tumor biomarkers including PD-L1 and PD-L2, as well as NIR-fluorescent probes such as Cy7 to the platelet surface (Figures 7a,b; 14e), and the linking of nanoparticle reporters including Qdots, magnetic nanoparticles (Figure 14i,j,k) and nano-bubbles to the platelet surface. Finally nanoparticles, including magnetic nanoparticles and structured gold including SPR-responsive nanogold, can be loaded via endocytosis or sequestration in KabC -platelets. These probes and nanoparticles are used to generate high-contrast in vivo signals from theranostic platelets that are used to image at their target tumor cells within the body. In the case of magnetic nanoparticles the application of an external magnetic field can be used to direct the platelet to the affected diseased tissue while higher magnetic field strengths can be used to heat the nanoparticle in the platelet and release the cytotoxic cargo to the targeted tumor.

[0059] Thrombin- or ADP-mediated platelet activation results in a host of protein processing and biosynthesis from resident mRNAs that trigger in a different set of interactions with immune and tumor cells while sub-platelet particles are linked to angiogenesis and binding to metastatic tumor cells, especially those in the blood where they afford protection against the immune system and the ravages of fluid forces. We have seen how supplementing natural interactions between un-activated platelets and tumor cells growth with transferrin can be exploited for tumor detection and drug delivery. We also propose using sub-activated platelet particles (SaPPs) as vehicles for theranostic agents. The new "activation-triggered" ligands on activated SaPP may result in different sets of interactions of the SaPP with tumor cells and immune cells that improve their targeting, drug delivery and in bridging tumor cells to secondary targets including T-cells and NK-cells.

[0060] Activated intact platelets are viewed as unsuitable vehicles for tumor targeting, owing to protrusions and tendency to aggregate. In some embodiments, SaPPs, are generated from fresh platelets activated with 10 mM ADP or iU/ml of thrombin and then subjected to sonication as described for unactivated nano-platelets. The SaPPs can be loaded with drugs, detection probes and surface-borne targeting groups, either in the original unactivated platelet or after sonication.

[0061] As the theranostic platelets and nano-platelets injected into the patient are derived from that patient's blood, the theranostic approach is referred to as an "arm back to arm" therapy.

[0062] Aspects of the present disclosure include the design, preparation, characterization and applications of human platelets and their ultrasound-generated nanoscale sonicates (nano- platelets) as theranostic agents for imaging tumors, and for the targeting and delivery of cytotoxic drugs to tumor cells. While the examples and procedures provided in the following sections refer to human platelets and their sub-fragments re-configured to function as theranostic agents for in vivo and in vitro imaging, targeting and delivery of cytotoxins and for magnetic and optical heating of theranostic platelets bound to their target tumor cells, it is understood that other compositions including human red blood cells and their sub-fragments, and other fractions of human cells can be reconfigured to serve as theranostic agents to image and treat different types of human disease.

[0063] The first and most important step in preparing theranostic platelets is to inhibit thrombin- and other effector molecule-mediated activation of platelets. This feature is realized by treating purified platelets with a small molecule membrane permeable drug that binds to and inhibits actin polymerization in the cytosol of the platelet (Figure 3A,B; 14b,c). Additional methods to inactivate platelet activation include treatments with aspirin (or salicylic acid) to permanently inactivate cyclo-oxygenase (COX) in the platelet enzyme prostaglandin H- synthase, or adding prostaglandin, a well know inhibitor of platelet activation. Theranostic platelets are understood to harbor any number of probes that generate high contrast signals for in vivo imaging of their distribution including NIR fluorophores, luciferases and artificial luciferases, and nanoparticles and polymers that intrinsically or naturally generate signals that can be detected and manipulated by a magnetic field, NIR-fluorescence, MRI, electron spin resonance, NIR-absorption and light-scattering, surface plasmon resonance (SPR), ultrasound and radioactivity. These probes may be loaded separately, or in specific combinations, to the cytosol of the platelet by passive diffusion or endocytosis or sequestration, or by being genetically-encoded via a transfected mRNA or cDNA, or chemically-coupled to the surface of the platelet. The capture group may include ligands beyond the transferrin molecule shown herein that bind to targeted cells and include specific antibodies against tumor markers including PD-L1 and PD-L2 and their chemical conjugates with cytotoxic drugs or contrast agents, humanized antibodies, chemical and genetically engineered scaffolds that mimic antibodies in their specific binding to a target molecule, a sequence of a specific oligonucleotide or modified or unnatural oligonucleotide, RNAse, ribozymes, peptides, sugars and complex carbohydrates, lipids, liposomes, receptor-derived ligands, small molecule drugs and disruptors of protein- protein interactions including those associated with disruption of amyloid plaques, viral coat proteins and intact viruses, and genes encoding proteins including single chain therapeutic antibodies, HSP90 and endostatin (endostar) that once bound to a specific protein on the targeted cancer cell initiate apoptosis. The surface of the platelet or nano-platelet may also be co-labeled with the capture group and a ligand that on binding to a specific protein on the target cell induces endocytosis of the theranostic platelet or nano-platelet, folic acid or a basic arginine- or lysine-rich peptides that are taken across the platelet membrane via endocytosis-independent mechanisms. Theranostic platelets or nano-platelets are equipped with detection probes for in vivo imaging or quantification of their bound target. Detection probes include small molecule NIR-fluorescent, and fluorogenic probes that enter the platelet by passive diffusion and are trapped and become fluorescent after de-esterification by intracellular esterases. Also included are membrane permeable gadolinium chelates that are detected by the Tl relaxation time using MRI. The reporter may also include inorganic, organic or hybrid nanoparticle including those harboring iron oxide, NIR emitting dyes, (bio)-luminescent enzymes including lucif erases and artificial lucif erases, structured gold and other noble metals and electron spin resonance probes. The nanoparticle maybe internalized in the platelet via endocytosis or sequestration, or else chemically-coupled to the platelet via chemical reactions that link the nanoparticle to amino- or thiol-groups on the platelet surface.

[0064] Small cytotoxic drugs are loaded into the platelet or nano-platelet cytosol by passive diffusion, or else by forming pH-labile ester bonds between the drug and proteins on the outer surface of the platelet that are cleaved after endocytosis. Small molecule NIR activated photosensitizers including chlorin E6 are loaded passively to the platelet cytosol (Fig. 14f), or else chemically linked to the surface of the platelet. Large polymers or proteins including therapeutic antibodies, and proteins that induce apoptosis on binding to their target including Endostar and HSP90 are chemically-coupled to the outer surface of the platelet. Capture groups that recognize biomarkers on a target cell are introduced to the surface of the platelet using a mild coupling procedure (Figure 6,7, 14e,i). Other proteins and ligands including transferrin that on binding to the tumor cell induce endocytosis, are coupled to the outer surface of the platelet via a mild chemical reaction. Reconfigured theranostic platelets that are loaded by sequestration or endocytosis with magnetic nanoparticles (Fig. 14i,j,k) are directed to sites of disease including the brain by using an external magnetic field, whereas stronger magnetic fields are used to induce heating of nanoparticles at the platelet-target cell interface to bring about the release of cytotoxic agents that kill tumor cells. Relatedly, toxin release may be brought about by NIR optical-heating of gold nanoparticles, ultrasound-mediated heating of nanoparticles within theranostic platelets bound to, or endocytosed by targeted cells. Non-invasive release of cytotoxic cargo of theranostic platelets may also be triggered by NIR excitation of bound photosensitizers including chlorin E6 (Fig. 14f). Endocytosis of theranostic platelet and nano- platelets may release sequestered cytotoxic drugs directly, or after an acid mediated de- esterification of a pH-labile pro-drug within an endosome or lysosome. Blood platelets and nano-platelets repurposed as theranostic agents can target cells within brain tumors, or disrupt α/β-amyloid plaques linked to neurodegenerative diseases, including Alzheimer's and

Parkinson's.

[0065] In addition to small molecule probes and nanoparticles, platelets can also be transfected with cDNAs or mRNAs and mRNAs that encode proteins that serve as novel targeting agents for tumors, including a humanized form of a single chain antibodies that recognize specific epitopes on a mutated receptor protein in a cancer cell. The mRNA may also encode a detection protein including mRuby or related NIR fluorescent probe, or an MRI contrast agent including calcium- binding proteins such as parvalbumin that bind specifically to gadolinium that is taken up in the cytosol.

[0066] Aspects of the present disclosure include human platelets and their nanoscale sonicates with a cytosolic composition that includes KabC, aspirin (or salicylic acid or prostaglandin to inhibit platelet activation, cytotoxic drugs including doxorubicin. Also included are detection probes for in vivo imaging of theranostic platelets or nano-platelets that include cytosolic or surface-linked NIR emitting probes and Qdots, or gadolinium chelates for MRI. Photodynamic therapy agents including chlorin E6 may be introduced to the platelet cytosol bty passive diffusion and used as a probe for PTD or to release the cytotoxic drug after exposure to NIR. Additionally detection probes may include nanoparticles including fluorescent, magnetic, plasmonic or MRI contrast enhancing probes that are sequestered by KabC -platelets. Iron oxide nanoparticles may also be used to direct platelets to a tumor site via an external magnetic field. Theranostic platelets and nano-platelets may bear chemically-linked capture groups including engineered antibodies or ligands on their outer surface that bind specifically to biomarkers on target cells. Theranostic platelets and nano-platelets may also contain a chemically linked protein such as transferrin on their outer surface to facilitate their endocytosis by target cells.

Definitions [0067] As used herein, the singular forms "a," "an," and "the" include the plural reference unless the context clearly dictates otherwise.

[0068] Reference to "about" a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to "about X" includes description of "X".

[0069] The term "subject" or "patient" is used synonymously herein to describe a mammal. Examples of a subject include a human or animal (including, but not limited to, a dog, cat, rodent, horse, sheep, cow, pig, goat, donkey, or rabbit).

[0070] The terms "treat," "treating," and "treatment" are used synonymously herein to refer to any action providing a benefit to a subject at risk for or afflicted with a disease state or condition, including improvement in the condition through lessening, inhibition, suppression, or elimination of at least one symptom, delay in progression of the disease, or inhibition of the disease.

[0071] The term "prophylactic administration" refers to any action in advance of the occurrence of disease to reduce the likelihood of that disease or any action to reduce the likelihood of the subsequent occurrence of disease in the subject.

[0072] The term "inactive platelet" as used herein refers to a platelet that cannot be activated by thrombin or ADP.

[0073] Activated platelets aggregate after exposure to thrombin or ADP in part due to a polymerization of actin subunits and formation of membrane protrusions. Thus, inactivated platelets do not aggregate in response to thrombin or ADP. Certain activation markers (for example, p-selectin) may appear on or in the inactivated platelet after thrombin or ADP treatment even though the platelets do not aggregate. For example, treatment of platelets by KabC or cytochalasin inactivates the platelets (and they do not aggregate in response to thrombin or ADP) even though the platelets may present an activation marker in response to thrombin or ADP. Accordingly, in some embodiments, inactivated platelets present an activation marker and do not aggregate in response to thrombin or ADP. In some embodiments, the inactivated platelets do not present an activation marker and do not aggregate in response to thrombin or ADP.

[0074] The term "nano-platelet" as used herein refers to a closed membrane fragment of a platelet. [0075] As used herein, the term "targeting group" is synonymous with the terms "targeting agent" and "capture group."

[0076] It is understood that aspects and variations of the invention described herein include "consisting" and/or "consisting essentially of aspects and variations.

[0077] The disclosures of all publications, patents, and patent applications referred to herein are each hereby incorporated herein by reference in their entireties.

EXEMPLARY EMBODIMENTS OF GENERATION OF INACTIVE PLATELETS

[0078] In some embodiments, theranostic platelets and nano-platelets are designed to bind to a targeted cell in the blood, in the lymph and in the micro-environments of tumor cells in any tissue, including the brain.

[0079] In some embodiments, theranostic platelets and nano-platelets are isolated from the patient being treated, which reduces rejection and the clearance rate compared to artificial delivery vehicles. This feature allows one to use lower dosage of the theranostic compared to a rapidly cleared vehicle, and since theranostic platelets have longer circulation times this will help to reduce the cytotoxin-mediated liver damage associated with shorter-lived nanoparticle theranostics.

[0080] In some embodiments, the procedures and reactions used to prepare imaging, therapeutic and theranostic agents from human platelets have been optimized to preserve their spherical shape and inactivated condition and are completed within one hour;

[0081] In some embodiments, platelets are inactivated soon after their purification. Activation brought about by thrombin or other molecular or cellular agents. Inhibition is achieved by inhibiting directly or indirectly actin polymerization using membrane permeable drugs not limited to kabiramide C (KabC) or latrunculin and where kabC is heron identified as the example. Cy5-fluorescence imaging and FACS analyses show that a fluorescent KabC

(Tetramethylrhodamine (TMR)-KabC) is readily permeable across the platelet membrane and retained in the cytosol at high concentration.

[0082] In some embodiments, TMR-KabC is used to quantify and to image KabC in the cytoplasm of blood platelets.

[0083] In some embodiments, KabC or related actin-binding drugs is used as a potent cytotoxin that can induce apoptosis once delivered to the cytoplasm of a target cancer cell. [0084] In some embodiments, KabC, and fluorescent derivatives and other agents that block platelet activation are used to stabilize platelets for a period of 1 week without any damage or evidence of activation

[0085] In some embodiments, platelet inactivation is realized by treating purified blood platelets periodically with aspirin (or salicylic acid) or related inhibitor of the COX enzyme of prostaglandin H-synthase

[0086] In some embodiments, platelet inactivation is realized by treating purified blood platelets periodically with aspirin (or salicylic acid) or Prostaglandin (500 nM).

[0087] In some embodiments, platelets are inactivated by any one or more of KabC, cytochalasin, aspirin (or salicylic acid), or prostaglandin E.

EXEMPLARY EMBODIMENTS OF PASSIVE LOADING OF DRUGS, PROBES AND NANOPARTICLES

[0088] In some embodiments, platelets are loaded with a cytotoxic drug not limited to doxyrubicin and other small molecule, cell permeable agents that act in the targeted cell to bring about their death. Doxorubicin is retained in the cytosol of platelets as shown by FACS (Fig. 5).

[0089] In some embodiments, fluorogenic probes not limited to fluorescein di-ester and related fluorogenic NIR fluorogenic di-esters are taken up by platelets including KabC treated platelets and de-esterified in the cytosol and rendered fluorescent for in vitro and in vivo imaging theranostic platelets (Fig. 5).

[0090] In some embodiments, membrane permeable fluorescent probes not limited to hydrophobic NIR dyes and probes used for photodynamic therapy (PDT), including chlorin E6 are taken up by platelets including KabC-platelets and used for NIR-imaging of their fluorescence (Fig. 14,21) and as a PDT agent to treat of tumors, or to release cytotoxins from the platelet cytosol after exposure to NIR light.

[0091] In some embodiments, cell permeable sensors of reactive oxygen species (ROS) including di-hydrocyanines derived from Cy7, Cy 5.5, Cy5, and uncharged forms of these probes are used in platelets including KabC-platelets as fluorogenic NIR fluorescent sensors of ROS, which are known to be produced in tumors and at sites of infection, inflammation and injury. [0092] In some embodiments, platelets including KabC-platelets are loaded with cell permeable sensors of reactive oxygen species (ROS) are used to serve as sensors of ROS that are produced in tumors and at sites of infection, inflammation and injury.

[0093] In some embodiments, platelets including KabC-Platelets are loaded with cell permeable gadolinium chelates such as Magnevist and related MRI probes not limited to manganese chelates that serve as contrast enhancing probes for MRI (Fig. 14,18).

[0094] In some embodiments, platelets including KabC-Platelets are loaded with nanoparticles including magnetic, structured gold, polymers, nano-bubbles by endocytosis or sequestration with image contrast enhancing properties not limited to detection and imaging based on NIR- fluorescence, MRI, SPR, and ultrasound (Fig. 14, 18).

EXEMPLARY EMBODIMENTS OF CHEMICAL-LINKING OF BIOMOLECULES TO THE SURFACE OF THE PLATELET

[0095] In some embodiments, a capture group (targeting group) is composed of a protein, such transferrin or antibodies directed against tumor antigens and their conjugates, chemical or genetically-engineered scaffolds that mimic antibodies in their binding to a target antigen, a sequence of a specific oligonucleotide or modified or unnatural oligonucleotide, RNAse, ribozymes, peptides, sugars or complex carbohydrates, lipids, liposomes, small and larger molecule ligands, ligands that bind to receptors on the target cell, including small molecule and protein based disruptors of protein-protein interactions associated with disruption of amyloid plaques is introduced by specific chemical coupling to the external surface of the platelet.

[0096] In some embodiments, a protein derived capture group is attached covalently to the surface of a platelet or KabC-platelet via amino groups on lysine residues on membrane associated proteins using a NHS-ester or related amino reactive functional group that is present on a NIR-fluorophore, or a photosensitizer, or a MRI, probe including gadolinium chelates, or a defined DNA sequence, or photodynamic therapy probe, or a nanoparticle including those composed of biopolymers, liposomes, nano-bubbles structured gold- and iron oxide.

[0097] In some embodiments, platelets or KabC-platelets are treated with iminothiolane, or a related reagent that reacts with free amino groups on proteins on the outer face of the platelet membrane to generate free thiol groups or related functional groups that are then used to react with an appropriate reactive group including maleimides, acrylates or oxiranes on a targeting agent, drug, sensor probe or drug. [0098] In some embodiments, platelets or KabC-platelets are treated with a hetero-bifunctional crosslinking reagent that generates functional groups that are used for orthogonal coupling of a targeting group to the surface of the KabC-platelet including reactions with amino, thiol, alcohol, aldehydes, phenolic or other group naturally present or chemically or enzymatically generated on the surface of the surface of the platelet. These functional groups are then used to form a covalent bond with an appropriate reactive group that is linked to the targeting agent or detection probe or therapeutic agent.

[0099] In some embodiments, platelets or KabC-platelets are treated with a hetero- bifunctionalized polyethyleneglycol, or related biocompatible agent that reacts with free amino groups on proteins on the outer face of the platelet membrane to generate free maleimide groups, thiols, alkynes, azides, or any functional group that can be used to chemically-link a targeting agent bearing an appropriate reactive group

[0100] In some embodiments, maleimido-benzoic acid N-Hydroxysuccinimide ester (MBS) or a PEG-based hetero-bifunctional crosslinking reagent is reacted with amino groups on lysine residues of transferrin, antibodies or functionalized nanoparticles including those that serve as reporter probes for MRI, NIR- absorption, SPR, ultrasound, NIR-fluorescence, bioluminescence, radioactive emitters or nano-bubble to generate free maleimide group, thiol, alkyne, azide, or any functional groups that are used to chemically-link the targeting agent or probe to the surface of platelets or KabC platelets treated with an orthogonal functional group

[0101] In some embodiments, iminothiolane-treated platelets or KabC-platelets are reacted with MBS conjugates of an antibody that recognizes a specific tumor biomarker, including PD- Ll and PD-L2, or transferrin or a related ligand that binds to a protein that is over-expressed on the tumor cell, a specific sequence of DNA or related nucleic acid polymer, a reporter nanoparticle or a ligand or small molecule reporter probe or nanoparticle or nano-bubble, where in each case a covalent bond is formed between the maleimide group on the targeting group and a thiolated-protein on the outer surface of the platelet.

[0102] In some embodiments, iminothiolane treated platelets or KabC-platelets are co-reacted with an MBS conjugate of a targeting ligand or antibody, and a MBS-conjugate of a detection probe, or an MBS-conjugate of a protein that initiates apoptosis on binding to the tumor biomarker. [0103] In some embodiments, fluorescence imaging and FACS analysis are used to quantify the fluorescence labeling of a capture protein on the theranostic platelet and to quantify the binding, endocytosis and fate of the theranostic platelet on the target cancer cell.

EXEMPLARY EMBODIMENTS OF TRANS FECTIQN OF KABC-PLATELETS WITH cDNA and mRNA

[0104] In some embodiments, a cDNA or mRNA is transfected into human KabC-platelets that is transcribed by molecular machinery in the cytosol to produce a protein that serves as a ligand or as an engineered antibody that binds specifically to the biomarker on a diseased cell. Encoded genes are not limited to full length antibodies, and may include engineered humanized antibodies, antibody fusions with fluorescent proteins, such as mRuby, or capture groups or antibody fusions appended with tags that recruit a fluorescent or magnetic nanoparticle, Qdot or plasmonic gold nanoparticle.

[0105] In some embodiments, the capture group is encoded by a cDNA or mRNA that is transfected into the cytosol of KabC-platelets and translated by ribosomal machinery in the platelet cytosol to generate a targeting protein or humanized engineered antibody, a protein that on binding to its target induces apoptosis such as HSP90 or endostatin that is displayed on the external surface of the platelet or nano-platelet.

[0106] In some embodiments, a cDNA or mRNA is transfected into the KabC-platelet and translated by molecular machinery in the platelet cytosol to produce a fluorescent protein not limited to mRuby that serves as a near infra-red fluorescent reporter to image the platelet in vitro and in vivo or a protein that binds tightly to gadolinium including parvalbumin.

[0107] In some embodiments, a cDNA or mRNA that encodes a specific protein is transfected into KabC-platelets, and is translated and transcribed by the functional molecular machinery to produce a bioluminescence producing protein not limited to Renilla luciferase or an artificial luciferase that cleaves a specific substrate in the platelet cytosol to produce bio- or chemi- luminescence each serving as luminescent reporters for in vitro and in vivo imaging of the distribution of the theranostic platelet.

[0108] In some embodiments, a cDNA or mRNA is transfected into human platelets and translated by molecular machinery in the cytosol to produce an engineered protein that binds specifically to a biomolecule presented on the surface of a nanoparticle that is used to recruit the nanoparticle to the surface of the theranostic agent for imaging or therapy. EXEMPLARY EMBODIMENTS OF PRODUCTION OF ENGINEERED THERANQSTIC

PLATELETS FROM CYTOMEGAKARYOCYES

[0109] In some embodiments, a device is provided that carries out automatically the preparation of theranostic platelets and nano -platelets from a culture of cytomegakayrocytes.

[0110] In some embodiments, a device is provided that carries out automatically the preparation of theranostic platelets and nano -platelets from a culture of cytomegakayrocytes transfected permanently with genes encoding a far red infrared fluorescent protein, apoptosis- inducing proteins such as endostatin and surface capture groups.

[0111] In some embodiments, different preparations of human cytomegakaryocyte are transfected with genes encoding membrane-directed humanized engineered proteins that in one case an antibody or protein ligand that binds to a target antigen on the cancer cell and in the second case a gene that encodes a reporter protein, or an activator of T-cells, or an antibody that sequesters an inhibitor of T-cell or NK-cell activation, or a gene encoding an activator of T-cell or NK-cell activation

EXEMPLARY EMBODIMENTS OF PRODUCTION OF THERANOSTIC NANO- PLATELETS

[0112] In some embodiments, nano-platelets are prepared from platelets or KabC-treated platelets labeled on their outer surface with a targeting protein, including transferrin or an antibody directed against a tumor marker. The breakage and resealing of platelet membranes by sonication (Fig. 15, 19) or other mechanical event, including extrusion through nanometer scale pores, produces sub-platelet particles (nano-platelets) that have an average diameter of 200 nm. Nano-platelets are produced with the targeting agent on their external face. Nano-platelets treated with KabC and with membrane permeable drugs not limited to doxorubicin or NIR- fluorescent sensors are shown to retain the targeting agent, sensor probes and drugs in their cytosol as quantified by using FACS and fluorescence imaging (Fig. 15).

[0113] In some embodiments, maleimide-benzoic acid N-Hydroxysuccinimide ester (MBS) or a PEG-based heterobifunctional reagent harboring an NHS -ester or a related amino-reactive group and a second reactive group not limited to a maleimide, is reacted with transferrin, antibodies or functionalized nanoparticles including those that serve as reporter probes for MRI, NIR- absorption, SPR, ultrasound, NIR fluorescence, bioluminescence, radioactive emitters or nano-bubble to form a conjugate that is then used to form a covalent bond between the conjugate and a platelet surface bearing an appropriate, orthogonal functional group not limited to free thiols

[0114] In some embodiments, iminothiolane-treated platelets or nano-platelets are reacted with the MBS conjugate of a targeting ligand, an engineered antibody directed against a target biomarker including PD-L1 and PD-L2, or transferrin or related ligand that binds to a protein that is over-expressed on the tumor cell, or a reporter nanoparticle or a ligand or small molecule reporter probe or nanoparticle or nano-bubble in order to form a covalent bond between the targeting agent to a protein on the outer surface of the platelet or nano-platelet pre-treated with iminothiolane.

[0115] In some embodiments, the imino-thiolane treated platelet or nano-platelet is co-reacted with an MBS conjugate of a targeting group, and a MBS-conjugate of a detection group, or MBS-conjugate of a protein that will trigger apoptosis in the target cell on binding to a receptor. A specific example would be iminothiolane treated platelets or nano-platelets bearing MBS conjugates of transferrin that binds specifically to any over-expressed transferrin receptor, and endostatin or endostar, which binds to nucleolin receptors on endothelial cells in newly forming blood vessels in the tumor and initiates their apoptosis.

[0116] In some embodiments, fluorescence imaging and FACS analysis are used to quantify the labeling of a probe or sensor to a platelet or nano-platelet and to quantify the binding, endocytosis and fate of the platelet to a target cancer cell.

[0117] In some embodiments, sonication of platelets or KabC-platelets bearing a surface bound Cy7-transferrin or a capture antibody against a target protein are sonicated with the resultant nano-platelets being re-loaded with KabC, and loaded with fluorescent probes and doxorubicin.

[0118] In some embodiments, platelets or nano-platelets are loaded with gadolinium chelates (Magnevist, texaphin or Gd-Chlorin E6 for MRI

[0119] In some embodiments, platelets or KabC-platelets linked on their surface with transferrin are loaded with nanoparticle probes are sonicated to generate nano-platelets bearing surface bound transferrin and entrapped nanoparticles that are subsequently loaded with membrane permeable doxorubicin and NIR-fluorescent probe molecules [0120] In some embodiments, platelets or KabC-platelets are transfected with a cDNA or mRNA that encodes a targeting protein or antibody or a detection probe followed by sonication to generate nano-platelets harboring the same encoded protein

[0121] In some embodiments, a device is provided that carries out automatically the preparation of theranostic nano-platelets from purified platelets.

EXEMPLARY EMBODIMENTS OF GENERATION OF ACTIVATABLE PLATELETS AND THEIR SUB-PLATELET PARTICLES

[0122] In some embodiments, platelets loaded in their cytosol with doxorubicin and a NIR detection probe are activated by the action of ADP or thrombin and processed to generate a population of SaPPs. SaPPs are chemically linked with transferrin or targeting antibody and reloaded with cytotoxin and detection probe and used as theranostic agents to image and treat tumors in any tissue, including the brain.

[0123] In some embodiments, SaPPs are chemically linked with transferrin or CD3e or related antigen and re-loaded with cytotoxin and detection probe and used as theranostic agents to image and treat tumors in any tissue, including the brain.

[0124] In some embodiments, platelets loaded in their cytosol with doxorubicin and harboring sequestered magnetic nanoparticles are activated by the action of ADP or thrombin and processed to generate a population of SaPPs. SaPPs are chemically linked with transferrin or targeting antibody and re-loaded with cytotoxin and detection probe and used as theranostic agents for MRI, magnetic-targeting and treatment of tumors.

EXEMPLARY EMBODIMENTS OF IN VIVO USES OF THERANOSTIC NANO- PLATELETS

[0125] In some embodiments, Cy5- and/or Cy7-transferrin labeled platelets or KabC-platelets or nano-platelets are added to tumor cells that over express the transferrin receptor and are seen to be taken up by via endocytosis to a far greater degree than platelets that lack the capture group, as seen by confocal fluorescence microscopy and FACS (Figures 7, 8; 141; 15i).

[0126] In some embodiments, theranostic platelets and nano-platelets harboring a Cy7 conjugated transferrin on their surface are imaged in a small animal NIR fluorescence imaging system (Caliper Instruments) and are shown to localize to tumors that develop from the transplantation of tumor cells (Figures 9,10,16,17;20). [0127] In some embodiments, excised organs from mice are imaged in the Caliper imaging system and are shown through their Cy7 fluorescence emission to localize to the tumor. NIR- fluorescence is also found in the liver and spleen, as part of the intrinsic clearance mechanism, and is also found in the excrements in the kidneys and urine; (Figures 9,10).

[0128] In some embodiments, theranostic platelets and nano-platelets and are shown from Cy7 imaging studies to circulate in mice for upto 1 week before being cleared by the liver and spleen (Figures 9,10; 20).

[0129] In some embodiments, theranostic platelets and nano-platelets are loaded with 40nm iron oxide nanoparticles and are used to image the distribution of the platelet using the T2 signal from MRI (Fig. 18b).

[0130] In some embodiments, theranostic platelets and nano-platelets are loaded with magnetic nanoparticles and directed to the tumor by applying an external magnetic field.

[0131] In some embodiments, theranostic platelets and nano-platelets are labeled with magnetic nanoparticles and are directed to the tumor by applying a magnetic field wherein a higher magnetic field is applied to heat the theranostic platelet releasing its cytotoxic cargo to the target cell.

[0132] In some embodiments, theranostic platelets and nano-platelets are labeled with gold nanoparticles and after a suitable time to allow for their binding to the tumor they are exposed to NIR radiation wherein optical heating of the gold nanoparticles in the theranostic platelet releasing its cytotoxic cargo to the target cell.

[0133] In some embodiments, theranostic platelets and nano-platelets are loaded or chemically labeled with contrast-enhancing nanoparticles for ultrasound imaging including endocytosed or surface linked nano-bubbles and after a suitable time to allow for their binding to the tumor they are exposed to a higher energy of ultrasound to heat the theranostic platelet releasing its cytotoxic cargo to the target cell.

[0134] In some embodiments, theranostic platelets and nano-platelets are loaded or labeled with NIR-activated photosensitive probes not limited to chlorin E6 and texaphin, or ROS- generating nanoparticles and after a suitable time to allow for their binding to the tumor they are exposed to NIR radiation wherein reactive oxygen species generated in the theranostic platelet are used to kill the target cell.

[0135] In some embodiments, platelets or nano-platelets are used to image and dissociate amyloid plaques that form in patients with AD. Thus theranostic platelets or nano-platelets loaded with an NIR probe, MRI using a Gd -chelate or magnetic nanoparticle are also labeled covalently on their surface with a small molecule or antibody-based disruptor of peptide interactions in α/β-amyloid plaques, to release individual peptides from aggregates associated with neurodegenerative disorders.

[0136] In some embodiments, a theranostic platelet loaded with nano-magnetic particles is co- labeled on its surface with an antibodies against amyloid plaques, and the AD=specific protease, nephrysin. A specific example of this co-labeled KabC-platelet is exemplified by iminothiolane treated KabC -platelets co-labeled with MBS conjugates of an antibody that binds specifically to α/β amyloid peptide in Alzheimer's disease (AD) plaques, and Neprilysin, a protease that specifically cleaves α/β amyloid peptide in AD plaques to help dissolve the plaque. The application of a focused magnetic field to the brain is used to localize these platelets whereupon they will bind to amyloid plaques allowing nephrysin to degrade amyloid peptides.

[0137] In some embodiments, theranostic platelets harboring loaded with magnetic

nanoparticles are also labeled on their surface with an antibody, or protein or cyclodextrin that binds to tightly cholesterol and is used to remove cholesterol from plaques that form on the walls of blood vessels.

ARM-BACK-TQ-ARM APPROACH TO DIAGNOSIS AND THERAPY

[0138] In some embodiments the cell based carrier may be a blood platelet or nano-platelet (from here on a reference to a platelet or a nano-platelet does not exclude one or other state), or a red blood cell or nanoscale derivative of the red blood cell or a fragment of any cell including but not limited to T cells isolated from the same patient or a donor that does not elicit an immune response in the patient.

[0139] In some embodiments, platelets obtained from a patient are re-engineered as theranostic platelets and injected into the same patient within a short period of time after drawing blood from that patient.

[0140] In some embodiments, a kit is provided that allows a person with technical skills to prepare theranostic platelets and nano-platelets.

[0141] Figure 1 illustrates the "arm back to arm" approach to diagnose and treat patients with cancer, cardiovascular and neurodegenerative diseases using their own platelets as the source of theranostic platelets. [0142] The protocols used to prepare theranostic platelets include: step 1, platelets are isolated from the blood of the patient to be treated with theranostic platelets.

[0143] In step 2 the platelets are treated with KabC to inhibit actin polymerization or else aspirin (or salicylic acid) or prostaglandin to block platelet activation during the preparation of theranostic platelets. A variation of step 2 involves loading KabC-platelets with CFDA or a related fluorogenic or membrane permeable NIR-fluorogenic probe that is de-esterified and trapped in the cytosol as the fluorescent di-anion. An additional variation involves loading KabC-platelets with a dihydrocyanine dye that on exposure to ROS generates a NIR-fluorescent probe that can be used to improve the contrast of imaging theranostic platelets at sites that are known to generate ROS including tumors, inflammation, stroke and infection. One further variation involves loading the KabC-platelet with a membrane permeable form of a gadolinium chelate, of which Magnevist is shown as a suitable example, although other Gd³⁺-chelates may also be used including Texaphin, and the Gd³⁺-complex of chlorin E6. Platelets loaded with Gd complexes serve as powerful probes for Tl -contrast enhanced MRI. Another variation is to load KabC-platelets with a detection nanoparticles, exploiting the intrinsic ability of platelets to sequester nanoparticles, including magnetic nanoparticles that serve as a means for MRI using the T2-relaxation time, NIR fluorescence-imaging of Qdots and dye coupled polymers, and SPR- imaging of structured gold nanoparticles. The magnetic nanoparticles also provide an opportunity to direct the theranostic platelet to a diseased site in the body by applying an external magnetic at that site.

[0144] In step 3 the platelet is loaded with a cytotoxin such as doxorubicin or a probe that is used to generate reactive oxygen species (ROS) including Chlorin E6.

[0145] In step 4 KabC-platelets loaded with detection probes are treated with iminothiolane at 39 mM, to generate free thiol groups.

[0146] In step 5 the thiolated platelets are treated with a capture group such as transferrin or an antibody against a disease biomarker that is co-labeled with Cy5- or Cy7, for in vivo imaging, and MBS whose free pendant maleimide groups are designed to react with thiol groups on the platelet surface. This latter step can be modified to include other capture groups including antibodies against surface markers on the target tumor cell and proteins such as endostatin that induce apoptosis when bound to cells harboring the nucleolin receptor. The entire procedure can be completed within 1 hour by one skilled in the art. DETAILED DESCRIPTION AND BEST MODE OF IMPLEMENTATION

[0147] Aspects of the present disclosure include a platelet composition comprised of freshly isolated suspension of human platelets that are treated sequentially as indicated in figure 1 with KabC or aspirin (or salicylic acid) or prostaglandin or related drug or treatment to inhibit their activation, followed by a membrane permeable fluorogenic probe to image the interior of the platelet, followed by doxorubicin which serves as a potent cytotoxin to trigger apoptosis in target cells, followed by iminothiolane to introduce thiol groups onto surface proteins of the platelet, and finally with a maleimide conjugate of a capture group and an endocytosis promoting agent that binds covalently via the iminothiolane generated thiol groups on the surface of the platelet. These theranostic platelets and their nano-platelet derived fragments are designed to bind to and to kill tumor cells. Also provided are extensions to the methods and practices to produce theranostic nano-platelets. Also provided are extensions of the methods and practices to introduce additional targeting, delivery and imaging agents to theranostic platelets and nano- platelets including magnetic nanoparticles that are loaded by endocytosis or sequestration or by chemical coupling to the surface of the theranostic platelet and are used to direct the theranostic platelets to the target tissue via a magnetic field, and then to initiate local heating of the theranostic platelet bound to their target cell by increasing the amplitude of the magnetic field which releases the cytotoxic cargo. Also provided are methods to load other nanoparticles to the theranostic platelet and nano-platelets including nano-gold, NIR-fluorescent quantum dots, organic polymers labeled with contrast agents and ultrasound and MRI contrast enhancing agents. Also provided are methods to release ROS from platelets via NIR irradiation of chlorin E6 (Fig. 14f) or related NIR-activated photodynamic therapy probe or a photosensitizing NP. Also provided are methods to prepare theranostic red blood cells and nanoscale red blood cells, and other cell fragments isolated from the patient.

[0148] Ideally one would like to develop a rapid and efficient procedure to carry out the various steps used to prepare theranostic platelets or nano-platelets that begin, as shown in figure 1, with the isolation of platelets from the patient to the final composition and form of the theranostic platelet, which would be injected into the same patient within 1-2 hours.

[0149] Also provided is an extension to the manual method of preparing theranostic platelets and nano-platelets to one where each step is carried out in a device that automates the loading, reactions and ultrasound treatments of the original platelet within a microfluidic or milli-fluidic device. The output of the device is the final form of the theranostic platelet that is concentrated ready for injecting into the patient, as schematized in figure 11. Theranostic nano-platelets are produced in this device by including a specially constructed chamber that carries out the sonication (Fig 11).

[0150] An even more sophisticated version of this device would use a 3D chamber that supports the growth of human cytomegakaryocytes that produce platelets as shown in figure 12. The advantage of this system is that one could transfect cytomegakaryocytes with genes encoding an mRuby for in vivo imaging and a surface localized capture group that would be used to target the platelet to a target tumor cell. Theranostic nano-platelets are produced in this device by including a specially constructed chamber that carries out the sonication (Fig 12).

[0151] Aspects of the present disclosure include the design, optimization preparation, quantitative testing and analysis of theranostic platelets and nano-platelets directed against disease biomarkers. Also provided are extensions of the theranostic platelet and nano-platelet to red blood cells and nanoscale fragments of other cells.

[0152] Aspects of the present disclosure include the design, optimization preparation, quantitative testing and analysis of theranostic platelets and nano-platelets that harbor mRNAs or cDNAs or mRNAs that can be released into the cytosol of a targeted cell to produce a protein product that corrects a genetic-defect not limited to the production of the CFTR in the lung epithelia of patients with cystic fibrosis and other diseases where dysfunction result from the production of a mutated protein that can be corrected with the functional form of that protein. The means to deliver the mRNA or cDNA that encodes the correct form of the protein within or on the surface of the theranostic platelet are many fold and not limited to a theranostic platelet harboring a targeting group to direct the platelet to the lung epithelia (in the case of CFTR). Release of the intact cDNA or mRNA to the cytosol may be effected through several known mechanisms including the co-linkage of a lipase or streptolysin O to the outer surface of the platelet to effect the break down of the endosomal membrane or by including nona- arginine peptides on the surface of the platelet or nanoplatelet to trigger their passage across the plasma membrane via a non-endocytotic mechanism.

DIAGNOSTIC COMPOSITIONS

[0153] Aspects of the present disclosure include theranostic platelet and nano-platelet compositions that include blood platelets isolated from the patient, a chemically coupled-capture group (targeting group) that binds specifically to a biomarker on the targeted tumor cell, a cell permeable inhibitor of platelet activation that may take the form of small molecule inhibitor of actin polymerization in the platelet such as kabiramide C or aspirin (or salicylic acid) and prostaglandin that inhibit COX1 mediated signaling, membrane permeable cytotoxic drugs not limited to doxorubicin and detection probes not limited to fluorogenic esters that are trapped after de-esterification in the cytosol, nanoparticles including magnetic nanoparticles probes that are loaded via sequestration, or by chemical linkage of the sensor to the surface of the platelet. These probes and sensors are used for high-contrast in vivo imaging of theranostic platelets and to direct theranostic platelet to a target tissue by applying a localized magnetic field, or to release cytotoxins into the target cell or tissue by applying an external magnetic field that heats the nanoparticle and compromises the platelet membrane. The tumor targeting protein highlighted in this filing is transferrin, which binds tightly to transferrin receptors on the surface of tumor cells. The methods also allow for surface labeling of platelets with other targeting groups or combinations of capture groups not limited to antibodies directed against cell surface biomarkers on the tumor cell, endostatin or endostar and related proteins that trigger apoptosis in cells lining new capillaries in tumor cells, and unconventional capture agents including DNA, engineered antibodies and other scaffolds that can be used to bind specifically to a target group, cytotoxic drugs linked via pH labile chemical bond, and ligands that promote endocytosis or disruption of amyloid plaques.

[0154] Additional aspects of the present disclosure are illustrated in more detail in Figures 1- 21, filed herewith, the disclosures of each of which are herein incorporated by reference.

[0155] In certain embodiments, the disclosed theranostic platelet compositions are useful for the detection of tumors, delivery of cytotoxins to tumor cells, and in monitoringthe effectiveness of a drug treatment for cancer in a patient including tumors that form in the brain,

[0156] In certain embodiments, the disclosed theranostic nano-platelet compositions are useful for the detection, monitoring and delivery of cytotoxins to cancer cells in a patient including those in the brain, which are targetable because platelets are shown to cross the blood brain barrier.

[0157] In certain embodiments, the targeting group on the platelet or nano-platelet may include one or more ligand antibody and protease that allow the vehicle to bind to amyloid plaques or related prion like proteins associated with neurodegenerative disease including Alzheimer's, and to bring about their dissolution. [0158] "Patient" refers to human subjects. The terms "detection" and "imaging" as used herein means the quantification of tumor cells in a patient that includes measurements of signals from: a), small molecule probe including visible and NIR fluorescence and bioluminescence, contrast enhanced MRI, optical absorption, or b), signals from nanoparticle probes including those associated with NIR fluorescence and bioluminescence, magnetic field, ultrasound, surface plasmon resonance (SPR). The terms targeting agent, targeting protein and targeting ligand refer to a protein or other biomolecule that binds to a designated protein or biomolecule on the surface of a tumor cell, or to a designated protein or biomolecule in a diseased tissue including those biomolecules associated with AD and heart disease

METHODS OF ADMINISTRATION

[0159] The platelet and nano-platelet composition finds use for real time and non-invasive detection and diagnosis of cancer cells, or amyloid plaques in the body. The route of

administration may be selected according to a variety of factors including, but not limited to, the actual health condition, the formulation and/or device used to administer the treatment, and the condition of the patient to be treated. Routes of administration useful in the disclosed methods include but are not limited to injection into the blood stream or interstitial space around the tumor or injection of theranostic platelets that are trapped within a temperature sensitive hydrogel that is injected in its liquid state into the patient and forms in the vicinity of the tumor (Figure 13).

[0160] The period of time a subject is exposed to a theranostic platelet may depend, at least, on the particular tumor, and severity of the disease. The diagnostically effective time of exposure of the target cells to particular levels of theranostic platelet or nano-platelet can be determined by one who is skilled in the art, with a goal of achieving a statistically significant result for one or more compositions of the theranostic platelet.

[0161] Embodiments of the present disclosure also include combinations of one or more disclosed targeting groups with one or more other cytotoxins or sensor probe whose signals correlate to specific diseased states.

METHODS OF DIAGNOSIS

[0162] The theranostic compositions are useful for real-time, in vivo detection and evaluation of tumors or diseased tissue in a subject and the effectiveness of the cytotoxin cargo and associated toxic effects of the theranostic composition in killing tumor cells. Accordingly, the present disclosure provides methods of the targeting of platelets and their nanoscale fragments to tumors and tumor cells, with the chemical labeling or loading of platelets and nanoplatelets with organic or inorganic based sensors, the imaging and detection of platelets using NIR

fluorescence or other optical means, ultrasound and MRI, and the delivery of cytotoxins to tumor cells by administering a theranostic platelet or nano-platelet composition. For example, the present disclosure provides a method of imaging and quantifying cancer cells in defined tumors in a small animal and in determining the effectiveness of the drug therapy through in vitro and in vivo imaging of platelet probes within targeted tumor cells using small molecule sensors and nanoparticle probes including those that generate contrast by changes in signals associated with NIR-fluorescence or bioluminescence, MRI, ultrasound, magnetic fields or surface plasmons.

EXAMPLES

[0163] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the embodiments, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. As will be understood, by those of skill in the art of biochemistry, organic synthesis, cell biology, tumor biology and medicinal chemistry the specific conditions stated below can be varied or adapted to other reagents and products. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius with room temperature being that typical for a laboratory environment.

[0164] Additional examples of the present disclosure are described in more detail in Figures 1- 21, filed herewith, the disclosures of each of which are incorporated by reference.

[0165] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the purpose, spirit and scope of the invention. In addition, modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the purpose, objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the present invention.

FURTHER EMBODIMENTS OF THE PRESENT INVENTION

[0166] A theranostic composition comprising:

[0167] Human or animal platelets; agents and protocols to prepare theranostic platelets and nano-platelets, wherein the intracellular cargo comprises of membrane permeable cytotoxic drugs, where KabC, aspirin (or salicylic acid) or prostaglandin is used to inhibit platelet activation (Figures 2 and 3) and doxorubicin or a related toxin is used to kill target cells, and endocytosed, sequestered or surface-linked nanoparticle including those that exhibit or respond to NIR-fluorescence and bioluminescence, MRI-contrast enhancement, ultrasound, magnetic fields and SPR for in vivo imaging, tracking of injected theranostic platelets, heating either optically or magnetically the platelet interior to release the cytotoxic cargo, and a targeting group including antibodies or ligands that are chemically-linked either with or without a detection probe to the surface of the platelet or via a recruited nanoparticle, or else encoded by a mRNA or cDNA that is transfected within the KabC-platelet, and used to direct the theranostic platelet or nano-platelet to a cancer cell or diseased tissue and to deliver the cytotoxic to the target cell.

[0168] The vehicle composition described herein, wherein the vehicle comprises of a purified suspension of un-activated platelets isolated from a patient or donor (Figure 1).

[0169] The platelet inactivation composition of described herein, wherein the cytosolic agent that inhibits platelet actin polymerization comprises of a membrane permeable KabC derivative or related cytotoxin that accumulates in the cytosol of the platelet (Figure 2,3) and maybe also labeled with a NIR fluorescent probe (Figures 2,3; 14a).

[0170] The platelet inactivation composition described herein, wherein the cytosolic agent that inhibits platelet activation is a membrane permeable KabC derivative or related cytotoxin that accumulates in the cytosol of the platelet (Figure 2,3) and maybe also labeled with a NIR fluorescent probe (Figures 2,3), or by adding aspirin (or salicylic acid), which modifies a specific residue on the COX1 enzyme to block signaling of platelet activation or else by adding prostaglandin to the same effect. [0171] The capture agent described herein, wherein the capture group comprises platelets whose surface is reacted with iminothiolane or related thiol generating reagent that is subsequently treated with a thiol reactive conjugate of the capture agent (Figure 6,7, 14e)

[0172] The nano-platelet composition described herein, wherein the vehicle comprises a sonicated preparation of a suspension of platelets loaded with KabC whose preparation is optimized to generate right-side-out sub-fragments of the original platelet (Figure 4; 15a-h).

[0173] The targeting composition described herein, wherein the linking of the capture agent to the surface of the platelet or nano-platelet comprises platelets modified at their surface with a probe that generates free thiol groups and their reaction with a thiol reactive conjugate of the capture agent.

[0174] The targeting composition described herein, wherein the capture group comprises platelets whose surface is reacted with hetero-bifunctional crosslinker not limited to classes of PEG terminated by a maleimide and NHS -ester, the latter reacting with protein derived amino groups on the surface of the platelet and the maleimide reacting with a capture group treated with iminothiolane or related thiol generating reagent.

COMPOSITIONS RELATED TO TARGETING GROUPS ON PLATELETS

[0175] The targeting composition described herein, wherein the capture group comprises platelets whose surface is reacted with heterobifunctional crosslinker not limited to classes of hetero-bifunctional PEG terminated by an NHS or maleimide that reacts with either the capture agent or the platelet surface, and azide, alkyne or related agents that reacts with via click chemistry to the appropriately orthogonally conjugated capture agent or platelet surface.

[0176] The targeting composition described herein, wherein the surface of the platelet comprises of a chemically linked capture groups including transferrin (Figure 6,7, 14e), an natural or engineered antibody against a cell surface biomarker on the target cell or another group that binds specifically to a molecule on the target cell or in the diseased tissue.

[0177] The targeting composition described herein, wherein the platelet or nano-platelet comprises surface linked or sequestered magnetic nanoparticles (Fig. 14j,k,i).

[0178] The targeting composition described herein, wherein engineered capture proteins on the surface of the platelet comprises cDNAs or mRNAs that are transfected into kabC-platelets and expressed as functional antibodies and other proteins for detection, targeting and/or therapy. EXEMPLARY COMPOSITIONS RELATED TO THE CYTQTQXINS IN TARGETED PLATELETS

[0179] The cytotoxic composition described herein, wherein the platelet or nano-platelet comprises of a cell permeable doxorubicin or related cytotoxin loaded in the cytosol of platelet or nano-platelet (Fig. 5a,14f)

[0180] The cytotoxic composition described herein, wherein the KabC-platelet or nano- platelet comprises of a cell permeable photodynamic therapy probe including chlorin E6 loaded in the cytosol of platelet or nano-platelet (Fig.l4f)

[0181] The cytotoxic composition described herein, wherein the KabC-platelet or nano- platelet comprises of a surface attached antibody, protein or ligand that on binding to the targeted cell initiates apoptosis.

[0182] The SaPP composition wherein a platelet with accumulated detection probe and cytotoxic drug is activated by ADP or thrombin and the SaPPs isolated and linked on their surface with a targeting protein for the tumor

[0183] The SaPP composition wherein a SaPP loaded with detection probe and cytotoxic drug is co-linked on its surface with tumor-targeting proteins including transferrin, and antibody that binds to an orthogonal tumor marker.

EXEMPLARY COMPOSITIONS RELATED TO THE IN VIVO DETECTION OF TARGETED PLATELETS

[0184] The detection composition described herein, wherein the sensor is a membrane permeable NIR fluorescent derivative of KabC loaded in the cytosol of the kabC-platelet.

[0185] The detection composition described herein, wherein the distributions of theranostic platelets in the body comprises detection of NIR emission from NIR fluorescent probes loaded within or attached to the surface of the platelet (Figs. 9,10,16a;17a; 21c).

[0186] The detection composition described herein, wherein the kabC-platelet or nano-platelet comprises of a cell permeable or surface linked MRI contrast enhancing agent including

Magnevist (Fig. 14g,h, 18)

[0187] The detection composition described herein, wherein the kabC-platelet or nano-platelet comprises an endocytosed or sequestered or surface-linked NIR-fluorescent nanoparticle.

[0188] The detection composition described herein, wherein the kabC-platelet or nano-platelet comprises an endocytosed or sequestered or surface-linked gold nanoparticle. [0189] The detection composition described herein, wherein the platelet comprises an endocytosed or sequestered or surface-linked MRI-active nanoparticle (Fig. 14i,j,k, 18).

[0190] The detection composition described herein, wherein the platelet or nano-platelet comprises surface linked nano-bubbles.

[0191] The detection composition described herein, wherein the platelet or nano-platelet comprises surface linked luciferases or their conjugates that generate light via a

bioluminescence.

[0192] The detection composition described herein, wherein detection probes comprises cDNAs or mRNAs encoded NIR-fluorescent proteins that are transfected into purified platelets.

[0193] The detection composition described herein, wherein the platelet or nano-platelet comprises mRNA or cDNA encoded, or surface-linked, luciferase enzymes that generate light from catalytic turnover of a luciferin or chemi-luminescent substrate.

[0194] The detection composition described herein, wherein the distributions of theranostic platelets in the body comprises detection of the NIR emission from a photodynamics therapy probe including chlorin E6 loaded passively in the cytosol (Fig. 14f; 21c)

[0195] The detection composition described herein, wherein the distributions of theranostic platelets in the body comprises detection of NIR emission from a dihydrocyanine probe including dihydro-Cy7 loaded in the cytosol or linked to the platelet surface that is activated to a fluorescent Cy7-state by ROS generated at the site of the tumor or injury

[0196] The detection composition described herein, wherein the distribution of theranostic platelets in the body comprises detection of magnetic nanoparticles in the platelet.

[0197] The detection composition described herein, wherein the distribution of theranostic platelets in the body comprises x-ray detection of magnetic nanoparticles in the platelet.

[0198] The detection composition described herein, wherein the distributions of theranostic platelets in the body comprises MRI imaging of MRI contrast enhancing probes loaded within or linked to the surface of the KabC-platelet.

[0199] The detection composition of described herein, wherein the distributions of theranostic platelets in the body comprises ultrasound imaging of nano-bubble linked to the surface of the theranostic platelet.

[0200] The detection composition described herein, wherein the distribution of theranostic platelets in the body comprises NIR detection of plasmonic gold probes in the platelet. [0201] The detection composition described herein, wherein the distribution of theranostic platelets in the body comprises X-ray imaging of plasmonic gold probes in the platelet.

[0202] The detection composition described herein, wherein the distribution of theranostic platelets in the body comprises imaging bioluminescence from luciferases linked to the platelet surface.

[0203] The detection composition described herein, wherein the distribution of theranostic platelets in the body comprises imaging chemi-luminescence from substrates that are recognized by specific enzymes on platelets.

COMPOSITIONS RELATED TO THE DRUG DELIVERY OF THERANOSTIC-PLATELETS

[0204] The drug delivery composition described herein, wherein the location of the magnetic nanoparticle in the theranostic platelet or nano-platelet comprises control by an external magnetic field

[0205] The drug delivery composition described herein, wherein the release of cytotoxic drugs comprises magnetic field heating of nanoparticles in theranostic platelets at their target tissue.

[0206] The drug delivery composition described herein, wherein the platelet or nano-platelet comprises surface linked cytotoxins that are released from the platelet at the lower pH values found in the endosome.

[0207] The drug delivery composition described herein, wherein the release of cytotoxic drugs comprises heating theranostic platelets at their target tissue with NIR light.

[0208] The drug delivery described herein, wherein a surface linked protein on the platelet that is bound to or taken up by endocytosis in the target cell induces apoptosis (Fig. 8, 141; 15i)

[0209] The drug delivery described herein, wherein the surface of the platelet or nano-platelet comprises a non-protein agent linked at the surface of the platelet or nano-platelet that promotes endocytosis in the target cell (Fig. 141, 15i)

[0210] The drug delivery described herein, wherein the surface of the platelet or nano-platelet comprises drugs or enzymes that lyse the membrane of the endosome.

[0211] The drug delivery described herein, wherein target cell delivery of cytotoxins comprises surface- attached cytotoxic drugs that are released from the platelet or nano-platelet via pH sensitive hydrolysis of an ester bond [0212] The drug delivery composition described herein wherein endocytosis promoting proteins on the surface of the platelet comprises cDNAs or mRNAs or mRNAs that are transfected into purified platelets.

[0213] The cytotoxin composition described herein wherein apoptosis promoting proteins on the surface of the platelet comprises cDNAs or mRNAs encoding surface presented engineered therapeutic antibodies or proteins including endostatin.

PROCEDURES RELATED TO THE METHOD OF PREPARING THERANOSTIC- PLATELETS

[0214] The theranostic platelet composition described herein wherein the procedures used to prepare theranostic platelet comprises a microfluidic device that carries out all of the small molecule loading and chemical attachment of capture groups and reporter probes on the surface of the platelet.

[0215] The theranostic platelet composition disclosed herein, wherein the procedures used to prepare theranostic nano-platelet comprises a microfluidic device that carries out sonication of intact platelets and all of the small probe and drug molecule loading and chemical attachment of capture groups and reporter probes on the surface of the nano-platelet (Fig. 11).

[0216] The heranostic platelet composition described herein wherein the procedures used to prepare theranostic nano-platelet comprises a microfluidic device that supports the growth of species specific cytomegakaryocytes transfected permanently with genes that encode the apoptosis inducing protein, mRuby and the capture protein and produce platelets and sonicated nano-platelets that are subsequently acted upon to load KabC and surface attached nanoparticle reporter probes as indicated in figure 12.

[0217] A method of preparing a theranostic platelet, the method comprising: isolating purified platelets from the blood of a patient, the composition comprising of a suspension of un-activated platelets in a neutral buffer wherein a stock solution of membrane permeable KabC is added as an inhibitor of actin polymerization in the platelet cytosol, wherein the platelet is treated with a stock solution of a potent cytotoxin or active agent, including doxorubicin that is retained within the cytosol of the platelet, wherein the platelet suspension is treated with a reporter probe or nanoparticle that is trapped in the platelet cytsosol after de-sterification, wherein the platelet is treated with iminothiolane to generate free thiol groups on the outer surface wherein a solution of a capture agent conjugated with a MBS crosslinker is used to form a covalent bond with the surface thiol groups.

[0218] The method described herein, wherein the platelet inactivating drug comprises of a cell permeable inhibitor of actin polymerization including KabC and related macrolides.

[0219] The method described herein, wherein the platelet inactivating drug comprises of aspirin (or salicylic acid) and/or prostaglandin.

[0220] The method described herein, wherein the cytotoxic drug comprises doxyrubucin or related cytotoxic drug.

[0221] The compound described herein, wherein the drug comprises a NIR-fluorescent derivative of kabC.

[0222] The compound described herein, wherein the cytotoxic drug in the theranostic platelet comprises a derivative with a pH labile bond that is cleaved in the endosome of the target cell to release the active drug.

[0223] The compound described herein, wherein the detection probe in the theranostic platelet comprises a membrane permeable MRI contrast-enhancing agent including gadolinium chelates including magnevist.

[0224] The nanoparticle detection probe described herein, wherein the probe comprises an endocytosed or sequestered nanoparticle including a magnetic nanoparticle, a gold nanoparticle, a NIR fluorescent nanoparticle.

[0225] The nanoparticle detection probe described herein wherein the probe comprises a surface linked nanoparticle including a magnetic nanoparticle, a gold nanoparticle, or a NIR fluorescent nanoparticle.

[0226] The nanoparticle detection probe described herein, wherein the probe comprises a nanoparticle including a ROS-sensing polymer or ROS generating polymer.

[0227] The detection probe described herein, wherein the probe comprises a cDNA encoding a fluorescent protein including mRuby that once transfected into the platelet will produce the cytosolic probe for NIR in vivo imaging of platelets

[0228] The targeting agent described herein, wherein the capture group comprises NIR coupled transferrin conjugates covalently linked to the surface of the platelet.

[0229] The targeting agent described herein, wherein the capture group comprises antibody conjugates against a target protein that is covalently linked to the surface of the platelet. [0230] The targeting agent described herein, wherein the probe comprises a cDNA that once transfected into the platelet will produce a membrane bound capture protein or engineered single chain antibody whose binding site for the target molecule is present on the outer surface of the platelet

[0231] The cytotoxic agent described herein, wherein the agent comprises a surface bound small molecule toxin that on binding to its target initiates apoptosis.

[0232] The cytotoxic agent described herein, wherein the agent comprises a surface bound protein that on binding to its target initiates apoptosis and includes antibodies, endostatin, Endostar and heat shock protein 90.

[0233] The therapeutic agent described herein, wherein the active agent comprises a small molecule including small molecule disruptors of amyloid plaques that on binding to the target plaque competes effectively in binding to /β amyloid peptides resulting in their removal from the plaque.

[0234] The therapeutic agent described herein, wherein the active agent comprises an antibody or protein that binds to α/β amyloid and on binding to a target plaque in the brain competes effectively in binding to α/β amyloid peptides resulting in their removal from the plaque.

[0235] The therapeutic agent described herein, wherein the active agents comprise an antibody or protein that binds to α/β amyloid and surface bound nephrilysin that on binding to a target plaque in the brain results in the proteolytic degradation of α/β amyloid peptides resulting in their removal from the plaque.

[0236] The therapeutic agent described herein, wherein the active agent comprises an antibody or protein or cyclodextrin that binds to cholesterol crystals and on binding to a target plaque on the vascular wall competes for interactions with cholesterol and leads to the removal of cholesterol crystals from the plaque.

[0237] The therapeutic agent described herein, wherein the active agent comprises a pH activated pro-drug that on encapsulation in the endosome of the target cell undergoes a pH- mediated de-esterification that releases the active form into the cytoplasm of the target cell.

[0238] The drug delivery vehicle described herein, wherein the targeting of the theranostic platelet comprises applying a magnetic field at the site of the tumor or amyloid plaque to attract the magnetic nanoparticle bearing theranostic vehicle. [0239] The drug delivery vehicle described herein, wherein drug release from the theranostic platelet comprises applying a stronger magnetic field to effect heating of the theranostic platelet at the site of the tumor or amyloid plaque to release the cytotoxic contents.

[0240] The method to produce theranostic platelets described herein, wherein the microfluidic device is comprised of a series of flow chambers wherein platelets isolated from the patient are loaded with kabC or aspirin (or salicylic acid) or prostaglandin, doxorubicin, NTR esters, MRI contrast enhancing agents nanoparticles, wherein and cDNAs or mRNAs are transfected, and wherein surface thiols are generated on the surface of the platelet and wherein the capture agent is chemically linked to the thiol groups on the platelet surface and wherein the theranostic platelets are concentrated.

[0241] The method to produce theranostic nano-platelets described herein, as schematized in figure 11 wherein the microfluidic device is comprised of a series of flow chambers as indicated in figure 11 wherein platelets isolated from the patient are loaded kabC wherein surface thiols are generated on the surface of the platelet and wherein the capture agent is chemically linked to the thiol groups on the platelet surface and wherein intact platelets, including intact platelets with magnetic, NIR fluorescent, gold nanoparticles are sonicated to produce nano-particles wherein doxorubicin, NIR esters are added and wherein the theranostic nano-platelets are concentrated.

[0242] The method of preparing a theranostic AaPP, the method comprising: as shown in the figure of isolating purified platelets from the blood of a patient, the composition comprising of a suspension of un-activated platelets in a neutral buffer the platelet is treated with a stock solution of a potent cytotoxin or active agent, including doxorubicin that is retained within the cytosol of the platelet, wherein the platelet suspension is treated with a reporter probe or nanoparticle that is trapped in the platelet cytsosol after de-sterification, wherein the platelet is treated with ADP or thrombin to induce activation and SPPs isolated by centrifugation. Also wherein the activated platelet is sonicated as described for theranostic nanoplatelets, The purified SaPPs are treated with iminothiolane to generate free thiol groups on the outer surface wherein a solution of a targeting agent including an antibody against a tumor biomarker or transferrin, previously conjugated with a MBS crosslinker, to form a covalent bond with the surface thiol groups on the SaPP. Other MBS-conjugates of targeting antibodies, enzymes or proteins may be co-linked to iminothiolane treated SaPPs for example an MBS-conjugate of a protease that can degrade amyloid proteins in AD plaques. METHODS RELATED TO THE USE OF THERANOSTIC PLATELETS

[0243] The method for using theranostic platelets and nano-platelets to treat tumors and other diseased state described herein wherein a preparation of theranostic platelets or nano-platelets with a surface borne capture group that detects a biomarker on the target cell or tissue to be treated is injected into the same patient from whom the original platelets were derived wherein the distributions of the theranostic agents are imaged by either optical, magnetic, plasmonic, ultrasonic or MRI instruments to determine the success of the agent in targeting the diseased tissue wherein after a suitable period of time the signals emanating from the theranostic agent are monitored to monitored to assess the success of the therapy that may include a reduction in tumor mass or dissolution of an amyloid plaque. Moreover, the signal from the theranostic agent is monitored in the urine and faeces.

[0244] The method of using theranostic platelets to treat tumors and other diseased states described herein wherein a suspension of the theranostic platelet or nano-platelet is mixed into a two state polymer including NIP AM and related temperature sensitive gelling system wherein the polymer-platelet mix is injected into the patient close to the site of the tumor or diseased tissue as indicated in figure 13 wherein over a period of days to weeks the stiffness of the NIP AM gel increases slowing the free diffusion of the theranostic platelets within the confines of the diseased tissue.

[0245] The method of using theranostic and nano-platelets platelets to treat tumors and other diseased states described herein wherein magnetic nanoparticles chemically linked to the surface of the theranostic platelet are injected into the patient and after a suitable period of time to allow for the theranostic agent to bind to target sites on tumor cells or on the diseased tissue the subject is exposed to a magnetic field at the site of the tumor or diseased tissue wherein the platelets will accumulate to increase the probability of a productive interaction with the target biomolecule on the diseased tissue.

[0246] The method of using theranostic and nano-platelets platelets to treat tumors and other diseased states described herein wherein magnetic nanoparticles chemically linked to the surface of the theranostic platelet are injected into the patient and after a suitable period of time to allow for the theranostic agent to bind to target sites on tumor cells or on the diseased tissue the subject is exposed to a high magnetic field wherein the platelets bound to the target cells will be heated by iron oxide nanoparticles thereby compromising the membrane of the target cell to bring about cell death.

[0247] The method of using theranostic and nano-platelets platelets to treat tumors and other diseased states described herein wherein gold nanoparticles chemically linked to the surface of the theranostic platelet are injected into the patient and after a suitable period of time to allow for the theranostic agent to bind to target sites on tumor cells or on the diseased tissue the subject is exposed to NIR radiation wherein the platelets bound to the target cells will be heated by absorption of the NIR light by the gold nanoparticle thereby compromising the membrane of the target cell to bring about cell death.

[0248] The method of using theranostic and nano-platelets platelets to treat tumors and other diseased states described herein wherein photodynamic therapy probes including Chlorin E6 (Fig. 14f; NIR photodynamic therapy probes or NIR sensitizing nanoparticles attached to the platelet membrane or trapped within the platelet cytosol are injected into the patient and after a suitable period of time to allow for the theranostic agent to bind to target sites on tumor cells or on the diseased tissue the subject is exposed to NIR radiation wherein the platelets bound to the target cells will be destroyed by oxygen radicals releasing their cytotoxic cargo into or around the target cell wherein nanoparticles thereby compromising the membrane of the target cell to bring about cell death

[0249] The method of using theranostic and nano-platelets platelets to treat tumors and other diseased states described herein wherein theranostic platelets are labeled with nanoparticles linked at their membranes or via endocytosis that act as agents for ultrasound imaging are injected into the patient and after a suitable period of time to allow for the theranostic agent to bind to target sites on tumor cells or on the diseased tissue wherein theranostic platelets bound to the target cells are imaged and then subjected to a higher power of ultrasound to compromise the platelet membrane and to release the cytotoxic cargo into and in the vicinity of the target cell.

[0250] The method of using theranostic and nano-platelets platelets to image tumors and other diseased states described herein wherein theranostic platelets are loaded either by using membrane permeable NIR dyes or endocytosed or surface linked NIR fluorescence emitting nanoparticles are injected into the patient and after a suitable period of time to allow for the theranostic agent to bind to target sites on tumor cells or on the diseased tissue wherein the platelets theranostic platelets bound to the target cells are made visible by recording the NIR emission. [0251] The method of using theranostic and nano-platelets platelets to image tumors and other diseased states described herein wherein theranostic platelets are linked at their membranes with red shifted luciferases or enzymes that catalyze reactions from red shifted chemi-luminescent substrates are injected into the patient and after a suitable period of time to allow for the theranostic agent to bind to target sites on tumor cells or on the diseased tissue wherein theranostic platelets bound to the target cells are made visible by recording the bioluminescence or chemi-luminescence in an instrument designed for that purpose.

[0252] The method of using theranostic and nano-platelets platelets to image tumors and other diseased states described herein wherein theranostic platelets are linked at their membranes with structured gold nanoparticles are injected into the patient and after a suitable period of time to allow for the theranostic agent to bind to target sites on tumor cells or on the diseased tissue wherein theranostic platelets bound to the target cells are detected and made visible by recording the surface plasmon resonance signal that is generated on exposure to NIR light using an instrument designed for that purpose.

[0253] The method of using theranostic and nano-platelets platelets to image tumors and other diseased states described herein wherein theranostic platelets are labeled with iron oxide nanoparticles linked at their membranes or via endocytosis or sequestration are injected into the patient and after a suitable period of time to allow for the theranostic agent to bind to target sites on tumor cells or on the diseased tissue wherein theranostic platelets bound to the target cells are detected and made visible by examining the patient in an MRI instrument.

[0254] The method of using theranostic and nano-platelets platelets to image tumors and other diseased states described herein wherein theranostic platelets loaded with or linked at their membranes with MRI contrast enhancing agents including gadolinium chelates or endocytosed nanoparticles are injected into the patient and after a suitable period of time to allow for the theranostic agent to bind to target sites on tumor cells or on the diseased tissue wherein theranostic platelets bound to the target cells are made visible by examining the patient in an MRI instrument.

[0255] The method of using theranostic and nano-platelets platelets to image tumors and other diseased states described herein wherein theranostic platelets are labeled with nanoparticles linked at their membranes or via endocytosis or sequestration that act as contrast-enhancing agents for ultrasound imaging are injected into the patient and after a suitable period of time to allow for the theranostic agent to bind to target sites on tumor cells or on the diseased tissue wherein theranostic platelets bound to the target cells are detected and made visible by examining the patient using a ultrasound instrument.

[0256] The method of using theranostic and nano-platelets platelets of described herein wherein the cytoxic drugs and detection probes or nanoparticles are localized to the interior of the theranostic platelet with only the linkage group of the targeting protein being recognized as foreign by the immune system

[0257] The method of using theranostic and nano-platelets platelets described herein for MRI- imaging wherein the MRI or NIR-fluorescent detection probes are localized to the interior of the theranostic platelet and recruitment of theranostic platelets is realized by the intrinsic binding of proteins on the platelet surface to cells in the tumor.

[0258] A kit comprising:

a theranostic platelet comprising:

blood platelets isolated from the patient that is reconfigured in a device to include capture groups specific for a biomarker on diseased cells, drugs to inhibit platelet activation, cytotoxic agents, reporter probes and targeting nanoparticles, wherein the platelet isolated from the patient is converted to the theranostic platelet within a short time and injected after sterilization into the same patient where after in vivo imaging of the reporter probe using the appropriate detection and imaging device to identify the site of the diseased tissue and monitor the progression of the therapy, and comprising magnetic nanoparticles to direct platelets to the site of action in the body and wherein a high magnetic field is applied to heat the nanoparticle and to release the cytotoxic contents of the theranostic platelet.

[0259] A kit comprising:

a theranostic nano-platelet comprising:

blood platelets isolated from the patient that is reconfigured in a device to include capture groups specific for a biomarker on diseased cells, drugs to inhibit platelet activation, cyotoxic agents, reporter probes and targeting nanoparticles, wherein the platelet isolated from the patient is converted to the theranostic nano-platelet within a short time and injected after sterilization into the same patient whereafter in vivo imaging of the reporter probe using the appropriate detection and imaging device to identify the site of the diseased tissue and monitor the progression of the therapy, and comprising magnetic nanoparticles to direct platelets to the site of action in the body and wherein a high magnetic field is applied to heat the nanoparticle and to release cytotoxins from theranostic nano-platelets. FURTHER EMBODIMENTS

[0260] Embodiment 1: A theranostic composition comprising:

Platelets; agents and protocols to prepare theranostic platelets and nano-platelets, wherein the intracellular cargo comprises of cytotoxic drugs, where KabC is used to inhibit platelet activation (Figures 2 and 3) and doxyrubicin or a related toxin is used to kill target cells, and an endocytosed nanoparticle including those related to NTR fluorescence and bioluminescence, MRI contrast enhancement, ultrasound, magnetic and SPR that is used for in vivo imaging and tracking of injected theranostic platelets, and a surface-bound capture group including those chemically linked to a reporter molecule or nanoparticle that is used to target the theranostic platelet or nano-platelet to a target cancer cell or tissue and to deliver the cytotoxic to the target cell.

[0261] Embodiment 2: The vehicle composition of Embodiment 1, wherein the vehicle comprises of a purified suspension of un-activated platelets isolated from a patient or donor

[0262] Embodiment 3: The platelet inactivation composition of Embodiment 1, wherein the cytosolic agent that inhibits platelet actin polymerization comprises of a membrane permeable KabC derivative or related cytotoxin that accumulates in the cytosol of the platelet (Figure 5) and maybe also labeled with a NTR fluorescent probe (Figures 6).

[0263] Embodiment 4: The targeting composition of Embodiment 1, wherein the surface of the platelet comprises of a chemically linked capture groups including transferrin (Figure 6 and7), an antibody against a cell surface biomarker on the target cell or another group that binds specifically to a molecule on the target cell

[0264] Embodiment 5: The nano-platelet composition of Embodiment 1, wherein the vehicle comprises a sonicated preparation of a suspension of platelets loaded with KabC whose preparation is optimized to generate right-side-out sub-fragments of the original platelet (Figure 4).

[0265] Embodiment 6: The imaging composition of Embodiment 1, wherein the sensor is a membrane permeable NIR fluorescent derivative of KabC.

[0266] Embodiment 7: The cytotoxic composition of Embodiment 1, wherein the platelet or nano-platelet comprises of a cell permeable doxyrubicin or related cytotoxin loaded in the cytosol of platelet of nano-platelet. [0267] Embodiment 8: The targeting composition of Embodiment 1, wherein the platelet or nano-platelet comprises an endocytosed magnetic nanoparticle.

[0268] Embodiment 9: The detection composition of Embodiment 1, wherein the platelet or nano-platelet comprises an endocytosed NIR fluorescent nanoparticle.

[0269] Embodiment 10: The detection composition of Embodiment 1, wherein the platelet or nano-platelet comprises an endocytosed gold nanoparticle.

[0270] Embodiment 12: The detection composition of Embodiment 1, wherein the platelet comprises an MRI contrast enhancing probe or endocytosed nanoparticle.

[0271] Embodiment 13: The detection composition of Embodiment 1, wherein the linking of the capture agent to the surface of the platelet or nano-platelet comprises platelets modified at their surface with a probe that generates free thiol groups and their reaction with a thiol reactive conjugate of the capture agent.

[0272] Embodiment 14: The targeting composition of Embodiment 1, wherein the platelet or nano-platelet comprises surface linked magnetic nanoparticles.

[0273] Embodiment 15: The detection composition of Embodiment 1, wherein the platelet or nano-platelet comprises surface linked NIR fluorescent nanoparticles.

[0274] Embodiment 16: The detection composition of Embodiment 1, wherein the platelet or nano-platelet comprises surface linked gold nanoparticles.

[0275] Embodiment 17: The detection composition of Embodiment 1, wherein the platelet or nano-platelet comprises surface linked luciferases or their conjugates that generate light via a bioluminescence.

[0276] Embodiment 18: The detection composition of Embodiment 1, wherein the platelet or nano-platelet comprises surface linked enzymes that generate light via from the turnover of a chemi-luminescent substrate.

[0277] Embodiment 19: The detection composition of Embodiment 1, wherein the platelet or nano-platelet comprises surface linked MRI contrast enhancing probe or endocytosed nanoparticle .

[0278] Embodiment 20: The detection composition of Embodiment 1, wherein the linking of the reporter probe to the surface of the platelet or nano-platelet comprises platelets modified at their surface with a probe that generates free thiol groups and their reaction with a thiol reactive conjugate of the reporter nanoparticle. [0279] Embodiment 21: The drug delivery composition of Embodiment 1, wherein the platelet or nano-platelet comprises surface linked cytotoxins that are released from the patelet at the lower pH values found in the endosome.

[0280] Embodiment 22: The detection composition of Embodiment 1, wherein the

distributions of theranostic platelets in the body comprises detection of NIR emission from NIR probes in the platelet.

[0281] Embodiment 23: The detection composition of Embodiment 1, wherein the distribution of theranostic platelets in the body comprises detection of magnetic nanoparticles in the platelet.

[0282] Embodiment 24: The imaging composition of Embodiment 1, wherein the distribution of theranostic platelets in the body comprises x-ray detection of magnetic nanoparticles in the platelet.

[0283] Embodiment 25: The detection composition of Embodiment 1, wherein the

distributions of theranostic platelets in the body comprises MRI imaging of MRI contrast enhancing probes in the platelet.

[0284] Embodiment 26: The detection composition of Embodiment 1, wherein the distribution of theranostic platelets in the body comprises NIR detection of plasmonic gold probes in the platelet.

[0285] Embodiment 27: The detection composition of Embodiment 1, wherein the distribution of theranostic platelets in the body comprises X-ray imaging of plasmonic gold probes in the platelet.

[0286] Embodiment 28: The detection composition of Embodiment 1, wherein the distribution of theranostic platelets in the body comprises imaging bioluminescence from luciferases on platelets.

[0287] Embodiment 29: The detection composition of Embodiment 1, wherein the distribution of theranostic platelets in the body comprises imaging chemi-luminescence from substrates that are recognized by specific enzymes on platelets.

[0288] Embodiment 30: The drug delivery composition of Embodiment 1, wherein the location of the magnetic nanoparticle in the theranostic platelet or nano-platelet comprises control by an external magnetic field

[0289] Embodiment 31 : The drug delivery composition of Embodiment 1 , wherein the release of cytotoxic drugs comprises magnetic field heating of nanoparticles in theranostic platelets at their target tissue. [0290] Embodiment 33: The drug delivery composition of Embodiment 1, wherein the release of cytotoxic drugs comprises heating theranostic platelets at their target tissue with NIR light.

[0291] Embodiment 34: The drug delivery of Embodiment 1, wherein promotion of endocytosis comprises a surface linked protein that is taken up by endocytosis in the target cell.

[0292] Embodiment 35: The drug delivery of Embodiment 1, wherein the surface of the platelet or nano-platelet comprises a non-protein agent linked at the surface of the platelet or nano-platelet that promotes endocytosis in the target cell.

[0293] Embodiment 36: The drug delivery of Embodiment 1, wherein the surface of the platelet or nano-platelet comprises drugs or enzymes that lyse the membrane of the endosome.

[0294] Embodiment 37: The drug delivery of Embodiment 1, wherein target cell delivery of cytotoxins comprises surface-attached cytotoxic drugs that are released from the platelet or nano-platelet via pH sensitive hydrolysis of an ester bond

[0295] Embodiment 38: The detection composition of Embodiment 1, wherein reporter probes comprises cDNAs that are transfected into purified platelets.

[0296] Embodiment 39: The targeting composition of Embodiment 1, wherein engineered capture proteins on the surface of the platelet comprises cDNAs that are transfected into purified platelets.

[0297] Embodiment 40: The drug delivery composition of Embodiment 1, wherein engineered endocytosis promoting proteins on the surface of the platelet comprises cDNAs that are transfected into purified platelets.

[0298] Embodiment 41: The cytotoxin composition of Embodiment 1, wherein apoptosis promoting promoting proteins on the surface of the platelet comprises cDNAs that are transfected into purified platelets.

[0299] Embodiment 42: A Theranostic platelet composition of Embodiment 1, wherein the procedures used to prepare theranostic platelet comprises a microfluidic device that carries out all of the small molecule loading and chemical attachment of capture groups and reporter probes on the surface of the platelet.

[0300] Embodiment 43: A Theranostic platelet composition of Embodiment 1, wherein the procedures used to prepare theranostic nano-platelet comprises a microfluidic device that carries out sonication of intact platelets and all of the small probe and drug molecule loading and chemical attachment of capture groups and reporter probes on the surface of the nano-platelet. [0301] Embodiment 44: A Theranostic platelet composition of Embodiment 1, wherein the procedures used to prepare theranostic nano-platelet comprises a microfluidic device that supports the growth of species specific cytomegakaryocytes transfected permanently with genes that encode the apoptosis inducing protein, mRuby and the capture protein and produce platelets and sonicated nano -platelets that are subsequently acted upon to load KabC and surface attached nanoparticle reporter probes as indicated in figure 12.

[0302] Embodiment 45: A method of preparing a theranostic platelet, the method comprising: isolating purified platelets from the blood of a patient, the composition comprising of a suspension of un-activated platelets in a neutral buffer wherein a stock solution of membrane permeable KabC is added as an inhibitor of actin polymerization in the platelet cytosol, wherein the platelet is treated with a stock solution of a potent cytotoxin or active agent, including doxyrubicin that is retained within the cytosol of the platelet, wherein the platelet suspension is treated with a reporter probe or nanoparticle that is trapped in the platelet cytsosol after de- sterification, wherein the platelet is treated with 1 mM iminothiolane to generate free thiol groups on the outer surface wherein a solution of a capture agent conjugated with a MBS crosslinker is used to form a covalent bond with the surface thiol groups.

[0303] Embodiment 46: The method of Embodiment 45 wherein the platelet inactivating drug comprises of a cell permeable inhibitor of actin polymerization including KabC and related macrolides.

[0304] Embodiment 47: The method of Embodiment 45, wherein the cytotoxic drug comprises doxyrubucin or related potent drug.

[0305] Embodiment 48: The compound of Embodiment 45, wherein the drug comprises a NIR derivative .

[0306] Embodiment 49: The compound of Embodiment 45, wherein the cytotoxic drug comprises a derivative with a pH labile bond that is cleaved in the endosome to release the active drug .

[0307] Embodiment 50: The compound of Embodiment 1, wherein the reporter probe comprises a membrane permeable MRI contrast-enhancing agent including gadolinium chelates.

[0308] Embodiment 51: The nanoparticle reporter probe of Embodiment 1, wherein the probe comprises an endocytosed nanoparticle including a magnetic nanoparticle, a gold nanoparticle, a NIR fluorescent nanoparticle. [0309] Embodiment 52: The nanoparticle reporter probe of Embodiment 1, wherein the probe comprises a surface linked nanoparticle including a magnetic nanoparticle, a gold nanoparticle, a NIR fluorescent nanoparticle.

[0310] Embodiment 53: The nanoparticle reporter probe of Embodiment 1, wherein the probe comprises a nanoparticle including a magnetic nanoparticle, a gold nanoparticle, a NIR fluorescent nanoparticle.

[0311] Embodiment 54: The reporter probe of Embodiment 1, wherein the probe comprises a cDNA encoding a fluorescent protein including mRuby that once transfected into the platelet will produce the cytosolic probe for NIR in vivo imaging of platelets

[0312] Embodiment 55: The capture agent of Embodiment 1, wherein the capture group comprises NIR coupled transferrin conjugates covalently linked to the surface of the platelet.

[0313] Embodiment 56: The capture agent of Embodiment 1, wherein the capture group comprises antibody conjugates against a target protein that is covalently linked to the surface of the platelet.

[0314] Embodiment 57: The capture agent of Embodiment 1, wherein the probe comprises a cDNA that once transfected into the platelet will produce a membrane bound capture protein whose binding site for the target molecule is present on the outer surface of the platelet

[0315] Embodiment 58: The cytotoxic agent of Embodiment 1, wherein the agent comprises a surface bound small molecule toxin that on binding to its target initiates apoptosis and includes antibodies, endostatin, Endostar and heat shock protein 90.

[0316] Embodiment 59: The cytotoxic agent of Embodiment 1, wherein the agent comprises a surface bound protein that on binding to its target initiates apoptosis and includes antibodies, endostatin, Endostar and heat shock protein 90.

[0317] Embodiment 60: The active agent of Embodiment 1, wherein the active agent comprises a small molecule including small molecule disruptors of amyloid plaques that on binding to the target plaque compete for interactions between the beta-sheets to disassemble the aggregate.

[0318] Embodiment 61: The active agent of Embodiment 1, wherein the active agent comprises a pH activated pro-drug that on encapsulation in the endosome of the target cell undergoes a pH-mediated de-sterification that releases the active form into the cytoplasm of the target cell. [0319] Embodiment 62: The targeting agent of Embodiment 1, wherein the targeting of the theranostic platelet comprises applying a magnetic field at the site of the tumor or amyloid plaque.

[0320] Embodiment 63: The drug delivery of Embodiment 1, wherein drug release from the theranostic platelet comprises applying a stronger magnetic field to effect heating of the theranostic platelet at the site of the tumor or amyloid plaque to release the cytotoxic contents.

[0321] Embodiment 64: The method of production of theranostic platelets of Embodiment 1, wherein the microfluidic device is comprised of a series of flow chambers wherein platelets isolated from the patient are loaded with kabC, doxyrubicin, NIR esters, MRI contrast enhancing agents nanoparticles, wherein and cDNAs are transfected, and wherein surface thiols are generated on the surface of the platelet and wherein the capture agent is chemically linked to the thiol groups on the platelet surface and wherein the theranostic platelets are concentrated.

[0322] Embodiment 65: The method of production of theranostic nano-platelets of

Embodiment 1, as schematized in figure 11 wherein the microfluidic device is comprised of a series of flow chambers as indicated in figure 11 wherein platelets isolated from the patient are loaded kabC wherein surface thiols are generated on the surface of the platelet and wherein the capture agent is chemically linked to the thiol groups on the platelet surface and wherein intact platelets, including intact platelets with magnetic, NIR fluorescent, gold nanoparticles are sonicated to produce nano-particles wherein doxyrubicin, NIR esters are added and wherein the theranostic nano-platelets are concentrated.

[0323] Embodiment 66: The method for using theranostic platelets and nano-platelets to treat tumors and other diseased state of Embodiment 1, wherein a preparation of theranostic platelets or nano-platelets with a surface borne capture group that detects a biomarker on the target cell or tissue to be treated is injected into the same patient from whom the original platelets were derived wherein the distributions of the theranostic agents are imaged by either optical, magnetic, plasmonic, ultrasonic or MRI instruments to determine the success of the agent in targeting the diseased tissue wherein after a suitable period of time the signals emanating from the theranostic agent are monitored to monitored to assess the success of the therapy that may include a reduction in tumor mass or dissolution of an amyloid plaque. Moreover, the signal from the theranostic agent is monitored in the urine and faeces.

[0324] Embodiment 67: The method of using theranostic platelets to treat tumors and other diseased states of Embodiment 1, wherein a suspension of the theranostic platelet or nano- platelet is mixed into a two state polymer including NIP AM and related temperature sensitive gelling system wherein the polymer-platelet mix is injected into the patient close to the site of the tumor or diseased tissue as indicated in figure 13 wherein over a period of days to weeks the stiffness of the NIP AM gel increases slowing the free diffusion of the theranostic platelets within the confines of the diseased tissue.

[0325] Embodiment 68: The method of using theranostic and nano-platelets platelets to treat tumors and other diseased states of Embodiment 1, wherein magnetic nanoparticles chemically linked to the surface of the theranostic platelet are injected into the patient and after a suitable period of time to allow for the theranostic agent to bind to target sites on tumor cells or on the diseased tissue the subject is exposed to a magnetic field at the site of the tumor or diseased tissue wherein the platelets will accumulate to increase the probability of a productive interaction with the target biomolecule on the diseased tissue.

[0326] Embodiment 69: The method of using theranostic and nano-platelets platelets to treat tumors and other diseased states of Embodiment 1, wherein magnetic nanoparticles chemically linked to the surface of the theranostic platelet are injected into the patient and after a suitable period of time to allow for the theranostic agent to bind to target sites on tumor cells or on the diseased tissue the subject is exposed to a high magnetic field wherein the platelets bound to the target cells will be heated by iron oxide nanoparticles thereby compromising the membrane of the target cell to bring about cell death.

[0327] Embodiment 70: The method of using theranostic and nano-platelets platelets to treat tumors and other diseased states of Embodiment 1, wherein gold nanoparticles chemically linked to the surface of the theranostic platelet are injected into the patient and after a suitable period of time to allow for the theranostic agent to bind to target sites on tumor cells or on the diseased tissue the subject is exposed to NIR radiation wherein the platelets bound to the target cells will be heated by absorption of the NIR light by the gold nanoparticle thereby

compromising the membrane of the target cell to bring about cell death.

[0328] Embodiment 71: The method of using theranostic and nano-platelets platelets to treat tumors and other diseased states of Embodiment 1, wherein NIR photosensitizer dyes or NIR sensitizing nanoparticles attached to the platelet membrane or trapped within the platelet cytosol are injected into the patient and after a suitable period of time to allow for the theranostic agent to bind to target sites on tumor cells or on the diseased tissue the subject is exposed to NIR radiation wherein the platelets bound to the target cells will be destroyed by oxygen radicals releasing their cytotoxic cargo into or around the target cell wherein nanoparticles thereby compromising the membrane of the target cell to bring about cell death

[0329] Embodiment 72: The method of using theranostic and nano-platelets platelets to treat tumors and other diseased states of Embodiment 1, wherein theranostic platelets are labeled with nanoparticles linked at their membranes or via endocytosis that act as agents for ultrasound imaging are injected into the patient and after a suitable period of time to allow for the theranostic agent to bind to target sites on tumor cells or on the diseased tissue wherein theranostic platelets bound to the target cells are imaged and then subjected to a higher power of ultrasound to compromise the platelet membrane and to release the cytotoxic cargo into and in the vicinity of the target cell.

[0330] Embodiment 73: The method of using theranostic and nano-platelets platelets to image tumors and other diseased states of Embodiment 1, wherein theranostic platelets are loaded either by using membrane permeable NIR dyes or endocytosed or surface linked NIR emitting nanoparticles are injected into the patient and after a suitable period of time to allow for the theranostic agent to bind to target sites on tumor cells or on the diseased tissue wherein the platelets theranostic platelets bound to the target cells are made visible by recording the NIR emission.

[0331] Embodiment 74: The method of using theranostic and nano-platelets platelets to image tumors and other diseased states of Embodiment 1, wherein theranostic platelets are linked at their membranes with red shifted luciferases or enzymes that catalyze reactions from red shifted chemi-luminescent substrates are injected into the patient and after a suitable period of time to allow for the theranostic agent to bind to target sites on tumor cells or on the diseased tissue wherein theranostic platelets bound to the target cells are made visible by recording the bioluminescence or chemi-luminescence in an instrument designed for that purpose.

[0332] Embodiment 75: The method of using theranostic and nano-platelets platelets to image tumors and other diseased states of Embodiment 1, wherein theranostic platelets are linked at their membranes with structured gold nanoparticles are injected into the patient and after a suitable period of time to allow for the theranostic agent to bind to target sites on tumor cells or on the diseased tissue wherein theranostic platelets bound to the target cells are detected and made visible by recording the surface plasmon resonance signal that is generated on exposure to NIR light using an instrument designed for that purpose. [0333] Embodiment 76: The method of using theranostic and nano-platelets platelets to image tumors and other diseased states of Embodiment 1, wherein theranostic platelets are labeled with iron oxide nanoparticles linked at their membranes or via endocytosis are injected into the patient and after a suitable period of time to allow for the theranostic agent to bind to target sites on tumor cells or on the diseased tissue wherein theranostic platelets bound to the target cells are detected and made visible by examining the patient in an MRI instrument.

[0334] Embodiment 77: The method of using theranostic and nano-platelets platelets to image tumors and other diseased states of Embodiment 1, wherein theranostic platelets loaded with or linked at their membranes with MRI contrast enhancing agents including gadolinium chelates or endocytosed nanoparticles are injected into the patient and after a suitable period of time to allow for the theranostic agent to bind to target sites on tumor cells or on the diseased tissue wherein theranostic platelets bound to the target cells are made visible by examining the patient in an MRI instrument.

[0335] Embodiment 78: The method of using theranostic and nano-platelets platelets to image tumors and other diseased states of Embodiment 1, wherein theranostic platelets are labeled with nanoparticles linked at their membranes or via endocytosis that act as contrast-enhancing agents for ultrasound imaging are injected into the patient and after a suitable period of time to allow for the theranostic agent to bind to target sites on tumor cells or on the diseased tissue wherein theranostic platelets bound to the target cells are detected and made visible by examining the patient using a ultrasound instrument.

[0336] Embodiment 79: A kit comprising:

a theranostic platelet comprising:

blood platelets isolated from the patient that is reconfigured in a device to include capture groups specific for a biomarker on diseased cells, drugs to inhibit platelet activation, cyotoxic agents, reporter probes and targeting nanoparticles, wherein the platelet isolated from the patient is converted to the theranostic platelet within a short time and injected after sterilization into the same patient whereafter in vivo imaging of the reporter probe using the appropriate detection and imaging device to identify the site of the diseased tissue and monitor the progression of the therapy, and comprising magnetic nanoparticles to direct platelets to the site of action in the body and wherein a high magnetic field is applied to heat the nanoparticle and to release the cytotoxic contents of the theranostic platelet.

[0337] Embodiment 80: A kit comprising: a theranostic nano-platelet comprising:

blood platelets isolated from the patient that is reconfigured in a device to include capture groups specific for a biomarker on diseased cells, drugs to inhibit platelet activation, cyotoxic agents, reporter probes and targeting nanoparticles, wherein the platelet isolated from the patient is converted to the theranostic nano-platelet within a short time and injected after sterilization into the same patient whereafter in vivo imaging of the reporter probe using the appropriate detection and imaging device to identify the site of the diseased tissue and monitor the progression of the therapy, and comprising magnetic nanoparticles to direct platelets to the site of action in the body and wherein a high magnetic field is applied to heat the nanoparticle and to release the cytotoxic contents of the theranostic nano-platelet.

EXAMPLE 1

[0338] The following protocols were used for the examples described below.

[0339] RPMI8226 multiple myeloma and K562 leukemia cells were obtained from the cell bank of the Chinese Academy of Sciences. Human platelets were obtained from the Chinese Red Cross within 5 days of drawing blood. Platelets were stored in citrate saline (0.006M tri-sodium

8 citrate/ 0.154M NaCl, pH 6.8) with 5% bovine serum albumin at a density of 3-5x10

platelets/ml. After centrifugation, platelet pellets were re-suspended in a modified Hanks' buffered salt solution (mHBSS; 0.17M NaCl/ 6.7mM KC1/ l.OmM MgS0₄/ 0.5mM K₂HP0₄/ 2.8mM Na₂HP0₄ / 13.8mM dextrose, pH to 7.2 with 1.4% NaHC0₃) for in vitro studies, or in normal saline for in vivo studies. KabC and TMR-KabC were prepared as described in Tanaka et al., Biomolecular mimicry in the actin cy to skeleton: mechanisms underlying the cytotoxicity of kabiramide C and related macrolides, Proc. Natl. Acad. Sci. USA, vol. 100(24), pp. 13851- 13856 (2003); Petchprayoon et al., Fluorescent kabiramides: new probes to quantify actin in vitro and in vivo, Bioconjug. Chem. vol. 16(6), pp. 1382-1389 (2005); and Pereira et al., Structural and Biochemical Studies of Actin in Complex with Synthetic Macrolide Tail

Analogues. Chem. Med. Chem., vol. 9(10), pp. 2286-2293 (2014). Human transferrin, EPI, carboxyfluorescein diacetate (CFDA), chlorin E6 (CE6), Phenyl-dimaleimide, maleimide- benzoic acid succinimide ester and iminothiolane were purchased from Sigma. Cy5-NHS and Cy7-NHS were both purchased from GE Healthcare.

[0340] Transferrin conjugates. N,N'-(l,4-phenylene)-dimaleimide (PDM)-transferrin was prepared by reacting 25 μΜ PBS solution of transferrin with 250 μΜ of 2-iminothiolane to generate thiol groups on the protein surface. After passage over a PD-10 column in PBS the protein fraction was treated with PDM to 250 μΜ and after a 2-hour incubation at 20°C the sample was applied to a second PD-10 column to remove excess crosslinker. The thiol-reactive conjugate was stored in 100 μΐ_^ aliquots at -20°C. A transferrin conjugate harboring both Cy5 and maleimide-benzoic acid succinimide ester (MBS) was prepared by incubating transferrin (5mg/ml) in PBS with Cy5-NHS (0.2mg/ml) and MBS (0.1 mg/ml) delivered from DMF stock solutions. After a 2-hour incubation unbound reactants were removed by PD-10 chromatography and the protein conjugate characterized by absorption spectroscopy and SDS-PAGE and stored at -20°C in ΙΟΟμί aliquots. The MBS/Cy7-conjugate of transferrin was prepared using the same protocol.

[0341] Confocal fluorescence microscopy: Fluorescence images of probes loaded into the cytoplasm or on the surface of platelets, and in mixtures of platelets with surface-attached RPMI8226 or K562 cells, within immuno-histocytochemical stained frozen myeloma xenograft tissue sections were carried out using a Zeiss 700 instrument with embedded software that allows for excitation of fluorescent probes (CFDA, FITC, EPI, TMR, or Cy5) at 488nm, 555nm and 639nm, and collection of the fluorescence emission from each probe was using a computer- selected filter.

[0342] FACS: A Becton and Dickenson FACSCalibur flow cytometer was used to sort and quantify labeled populations of free platelets and RPMI8226 or K562 cells. These studies recorded 10,000 events for each sample using the fluorescence of CFDA, FITC, EPI, TMR, Cy5 or CE6 that were detected using FL1, FL2, FL3 or FL4 channel respectively. The FACS data was analyzed with Flow Jo V3.2 (Tree Star, Inc.) and represented as the percentage of labeled population.

[0343] Electron microscopy: Platelets were prepared for SEM and TEM-imaging using an established protocol [27] in a Hitachi 450 SEM and a FEI Tecnai G2 Spirit Bio TWIN TEM. Suspensions of platelets were fixed chemically by adding an equal volume of 0.1%

glutaraldehyde for 15-minutes followed by centrifugation and re-suspension of the pellets in 3% glutaraldehyde. The platelet samples were allowed to settle and adhere to glass fragments that were pre-coated with poly-lysine. The glass fragments were rinsed with distilled water and dried at the critical-point.

[0344] Mice: Analgesic and tranquilizing drugs were used to minimize discomfort and pain to animals during injections of RPMI8226 cells. NOD/SCID mice are euthanatized by cervical dislocation after anesthetization with a 0.016 niL/g (body weight) solution of 2.5% Avertin that was injected intra-peritoneal. Alternatively, death was brought about by C0₂ anesthesia followed by decapitation.

[0345] NIR-fluorescence imaging: RPMI8226 cells (lxlO⁷ cells in 100 μΐ of buffer) were injected under the skin on the backs of NOD/SCID mice. In other studies RPMI8226 cells (lxlO⁶ cells in ΙΟμΙ) were injected through an opening in the skull of NOD/SCID mice.

Typically 3 mice were used in each data group. NIR fluorescence imaging of live mice and their excised organs was carried out using a Caliper IVIS Spectrum Imaging System. The instrument was used to record the NIR emission spectra of Cy5 and Cy7 loaded in platelets within live mice, or from their excised organs. The NIR fluorescence images shown in this study were processed using software resident in the IVIS machine. Prior to in vivo imaging, fur in the vicinity of the myeloma xenotransplant was removed from the mice by shaving or defoliation.

EXAMPLE 2: Suppressing specific and non-specific aggregation of human platelets

[0346] Platelet aggregation was inhibited by passive loading of freshly acquired, human platelets with a membrane permeable KabC or tetramethylrhodamine-KabC (TMR-KabC). Confocal fluorescence images of human platelets that had been incubated with TMR-KabC show the fluorescent drug accumulates in the cytoplasm, where it produces a strong emission that allows for high-contrast imaging of individual platelets (Figure 23A). The concentration dependence of TMR-KabC and KabC loading in platelets was further investigated further by measuring the TMR-fluorescence signal in populations of platelets as a function of incubation time and drug concentration (Figure 30). The final (standard) condition used to load KabC in platelets involved incubating -10 platelets with KabC (5μΜ) in mHBSS for 15-minutes at 22 C followed by centrifugation and washing with mHBSS. The standard KabC-loading condition resulted in 95.8% of platelets having a TMR- fluorescence signal that exceeded the highest fluorescence signal recorded for untreated platelets (Figure 23B). Once TMR-KabC or KabC crosses the plasma membrane, the compounds are effectively contained within the cytoplasm. Proof that TMR-KabC and KabC were effective in suppressing thrombin-mediated platelet aggregation was provided by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analyses of thrombin-treated platelets loaded with KabC (Figures 24A and 24B), or without KabC (Figures 24C and 24D). Platelets loaded with KabC maintained their characteristic smooth and spherical shape of the non-aggregated platelet after an exposure to thrombin (Figures 24 A and 24B), whereas platelets lacking KabC were irregular in shape, and displayed numerous membrane protrusions after a brief incubation with thrombin (Figures 24C and 24D).

EXAMPLE 3: Loading platelets with cytotoxins, NIR-fluorophores and photodynamic therapy probes

[0347] Having successfully inhibited platelet-aggregation using KabC, confocal fluorescence microscopy and FACS analyses were used to optimize conditions to load KabC -platelets with other membrane permeable drugs and detection probes. These drugs and probes included epidoxorubicin (EPI), a potent and intrinsically fluorescent cytotoxin (Figure 23C); carboxy- fluorescein di-ester, a fluorogenic probe used for in vitro fluorescence imaging (Figure 23D); and chlorin e6, a dual purpose, red-emitting fluorophore and photodynamic probe (Figures 23E and 23F). For example, FACS analysis showed that 97.9% of platelets incubated with 30 μΜ chlorin e6 for 15-minutes exhibited a far-red fluorescence, allowing the recording of high- contrast images of individual platelets during a single, 1- second scan of a field of view at a low power of 639 nm light. The fluorescence signals recorded for platelets loaded with each of the intrinsically fluorescent drugs/probes exceeded by a wide-margin the auto-fluorescence of unlabeled platelets (Figures 23E and 23F, vertical lines indicate background thresholds). FACS analysis was also used to show that KabC-platelets retained for at least 24-hours after their incubation with the membrane permeable probe (Figure 23E). Moreover, EPI even was retained within KabC-platelets after exposure to thrombin (99.6% versus 99.9%, respectively; Figures 2 and 2F), which suggests that KabC-platelets do not release their internalized cargo after thrombin treatment.

EXAMPLE 4: Surface-coupling of targeting proteins and NIR fluorescent probes

[0348] Targeting proteins and antibodies were chemically conjugated to the surface of KabC- platelets. Physiologically-relevant conditions were used for efficient coupling of human transferrin and tumor-targeting antibodies to the outer membrane of KabC-platelets. Transferrin was chosen to target RPMI8226 multiple myeloma and K562 leukemia, as previous studies have shown both cell types over-express the transferrin receptor. First, a 5mg/ml solution of a purified form of human transferrin (Sigma) was treated with an excess of 2-iminothiolane (Traut's reagent) in de-aerated PBS. The thiolated transferrin conjugate was isolated using PD- 10 chromatography in PBS, and subsequently mixed with an excess of N,N'-(l,4-phenylene)- dimaleimide (PDM) to generate pendant maleimide groups. Finally, the maleimide containing transferrin conjugate was incubated with a solution of Cy5-NHS or Cy7-NHS for 15-minutes to couple Cy5 or Cy7 molecules to lysine residues on the protein, which was followed by PD-10 chromatography. Transferrin conjugates bearing Cy5 and maleimide groups were concentrated and stored in 100 aliquots at -80°C.

[0349] Proof that Cy5 probes were chemically-coupled to transferrin was shown by SDS- PAGE analysis of the transferrin conjugate. The transferrin band (~80kD) was deliberately and massively over-loaded to visualize the Cy5-label in the unstained gel, which resulted in a blue- colored 80kD band owing to the absorption of red light by Cy5 molecules (Figures 25A and 25B). Absorption spectrometric analysis of the Cy5-transferrin conjugate revealed a labeling ratio of -4.5 (Cy5/transferrin; Figure 25C). The blue-shifted shoulder evident in the Cy5- absorption spectrum results from the stacking of Cy5 molecules on the transferrin molecule (Figure 25C), a phenomenon that reflects interactions among large aromatic molecules that takes place when multiple probes are attached to the same protein molecule.

[0350] Next, the doubly-labeled transferrin conjugate (Cy5/PDM) was coupled to KabC- platelets. KabC-platelets were first incubated with 2-iminothiolane (Traut's reagent) for 15- minutes in de-aerated buffer, which generated thiol groups on their outer membrane. These platelets were washed with de-aerated buffer, centrifuged at low speed, and re-suspended in a PBS solution containing Cy5/PDM-transferrin for 15-minutes. The chemical-coupling of PDM/Cy5-transferrin to KabC-platelets was confirmed using confocal fluorescence microscope imaging and FACS analysis (Figure 31). Cy5/transferrin-coupled KabC-platelets were imaged using bright-field and fluorescence confocal microscopy (Figure 25D) with the representative field showing single and spherical (i.e., non-aggregated) platelets that were homogenously labeled with Cy5. FACS analysis of a larger population of the Cy5/PDM-transferrin coupled KabC-platelets showed that 99.9% of platelets exhibited a Cy5-fluorescence intensity that exceeded the background emission recorded from unlabeled platelets (Figures 25E (background) and 25F (labeled), with the vertical lines indicating the background threshold; Figure 31). The high level of Cy7-labeling on platelets was also shown qualitatively from images of a gravity- sedimented suspension of Cy7-coupled KabC-platelets that absorb the far red region of the visible spectrum, and result in a visible blue sediment (Figure 31). [0351] In another method developed to chemically-link KabC-platelets with transferrin (or antibodies) and Cy5 (or Cy7), PDM-linked transferrin was incubated with thiolated KabC- platelets and, after washing and re-suspension, the coupled platelets were incubated with a 0.2 mg/ml solution of Cy5-NHS (or Cy7-NHS) for 15-minutes followed by centrifugation and re- suspension in mHBBS. The Cy5 or Cy7 -coupled KabC-platelets prepared by this method were used for studies are shown in Figures 26, 27, 28, and 32.

[0352] In another method developed to chemically-link KabC-platelets with transferrin (or antibodies) and Cy5 (or Cy7), thiolated KabC-platelets were coupled to a transferrin conjugate that had previously been doubly-labeled with Cy5-NHS and maleimidobenzoic acid succinimide acid (MBS). This method can be used to prepare platelets containing a cargo of cytotoxins, detection probes and surface-coupled targeting proteins within 90-minutes of receiving out-dated platelets.

[0353] Platelets prepared with any of these methods were found to be stable in buffer for 7- days or more. For example, the thrombin treated KabC-platelets shown in Figure 24A had been stored for 7-days at 4°C in platelet stabilizing buffer before being imaged in the electron microscope.

EXAMPLE 5: In vitro imaging of interactions between platelets and human tumor cells

[0354] Confocal fluorescence microscopy and FACS analysis was used to quantify interactions between Cy5/transferrin-coupled platelets and RPMI8226 cells and K562 cells. Each cell line was confirmed to over-express the transferrin receptor by incubating cells with a FITC-labeled antibody against the human transferrin receptor (Figures 26A and 26B) followed by FACS analysis. More than 70% of RPMI8226 cells, and more than 62% of K562 cells were found to have a FITC-fluorescence signals that exceeded that for unlabeled cells (indicated by the vertical lines in Figures 26A and 26B). Fluorescence images of KabC-platelets coupled with Cy5 or Cy5/transferrin were recorded following an 8-hour incubation with surface-attached RPMI8226 cells (Figures 26C, 26D, and 26E), or K562 cells (Figures 26F, 26G, and 26H). Visual inspections of the preparation in Cy5-fluorescence and bright-field modes showed most of the platelets were mobile, while the overlap of Cy5 and bright-field images showed KabC- platelets without transferrin did not bind to RPMI8226 cells or K562 cells (Figure 6C

(RPMI8226 cells) and 26F (K562 cells)). In contrast, a significant number of KabC-platelets coupled with transferrin and Cy5 were found to bind to RPMI8226 cells (Figures 26D and 6E) or K562 cells (Figures 26G and 26H). The discovery that surface-coupled transferrin could be used to promote KabC-platelet binding to RPMI8226 cells or K562 cells suggests that interactions between these tumor cells and platelets result from the interactions between transferrin on the platelet and transferrin receptors on the tumor cell. Neither RPMI8226 cells nor K562 cells showed obvious internalization of bound Cy5/transferrin coupled platelets, which would indicate the attached platelets are not endocytosed.

EXAMPLE 6: In vivo imaging of tumor-targeting platelets for a subcutaneous tumor

[0355] In vivo distributions of KabC -platelets surface-coupled with Cy7 and transferrin, or only with Cy7, were characterized in immuno-compromised mice. RPMI8226 cells were injected sub-cutaneously into the back of NOD/SCID mice. Myeloma xenotransplants were visible in mice around 2-4 weeks after injecting RPMI8226 cells. The average volume of xenografts that developed after subcutaneous injection of RPMI8226 cells reached 80-100 mm³ after 7-10 days, and increased to about 2000 mm³ by about 21 days.

[0356] A Caliper NIR-fluorescence imaging system was used to image and analyze the distributions of -10 KabC-platelets surface-coupled with Cy7 (control), or with Cy7 and transferrin (test). After allowing the xenografts to develop to - 100mm , test and control platelets were injected separately into the tail veins of NOD/SCID mice (n=3 per group), and whole body Cy7-fluorescence images recorded over a period of 6-days. Control and test platelets were found throughout the body of injected mice during the first 24-hours, as evidenced by the strong and uniform Cy7-fluorescence in representative mice (Figures 27A and 27B). KabC-platelets coupled with transferrin and Cy7 accumulated in sub-cutaneous xenografts that could be resolved by Cy7-fluorescence imaging against other fluorescent platelets within 96-hours of injection. The test platelets remained bound to cells in the sub-cutaneous tumor until at least day-6, after which time, they accounted for almost all of the Cy7 emission on the dorsal side of the animal (Figure 27B at 144 hours).

[0357] The targeting of platelets to myeloma xenografts that developed from injected RPMI8226 cells was evaluated from analyses of the Cy7-fluorescence signal in organs excised from mice injected with KabC-platelets coupled with Cy7 and transferrin (test), or Cy7-coupled KabC-platelets without transferrin (control) (Figure 27C). The area-normalized Cy7- fluorescence signals from excised tumors was 2.54 times higher for Cy7/transferrin-coupled KabC-platelets compared to Cy7-coupled KabC-platelets without transferrin (5.25 +/ -1.11 vs 2.07 +/- 1.12 (photons/sec/cm 2 /sr)/^W/cm 2 ) respectively). Cy7-fluorescence was also evident in the excised livers of mice injected with test or control platelets (13.32 +/- 2.13 and 16.85 +/-

1.78 (photons/sec/cm 2 /sr)/^W/cm 2 ), respectively), and in the spleens of mice injected with test and control platelets (1.89 +/- 0.99 and 2.41 +/-0.40 (photons/sec/cm²/sr) W/cm²), respectively). High levels of Cy7-fluorescence were also found in the kidneys of both groups of mice (2.79 +/- 1.37 and 4.33 +/- 1.49 (photons/sec/cm 2 /sr)/^W/cm 2 ), respectively), and in their urine. Given that most if not all chemical modifications of Cy7, including, oxidation or fragmentation, result in a loss of far-red absorption and fluorescence (λ_εχ ~650nm, λ_εχ ~670nm), the discovery that the urine of injected mice emitted a strong Cy7 fluorescence suggest the probe molecule was not modified during its residence in the mouse, and offers an opportunity to account for the fate of injected platelets.

[0358] The effectiveness of KabC-platelets coupled with transferrin (test) in targeting myeloma xenotransplants was evaluated by calculating the ratio of Cy7-fluorescence intensity among excised organs (myeloma xenotransplant, liver, spleen and kidney). The ratios for the tumor/liver, tumor/spleen and tumor/kidney in the test and control mice were consistently higher for test platelets compared to control platelets, and calculated as 0.37 vs 0.13, 2.71 vs 0.88, and 2.00 vs 0.61 respectively, with all p-values being less than 0.05.

EXAMPLE 7: In vivo imaging of tumor- targeting platelets for an intracranial tumor

[0359] Myeloma xenotransplants grew rapidly in the intra-cranial cavity, thus allowing injection of Cy7-labeled KabC-platelets into mice only 5-days after they had been transplanted intra-cranially with RPMI8226 cells. KabC-platelets coupled with Cy7 and transferrin were injected into the tail veins of control mice without RPMI8226 cells (Figure 28C; n=3; Figures 32A and 32B), and in mice that 5-days earlier had been injected intra-cranially with RPMI8226 cells (Figures 28A and 28B; n=3; Figure 23C).

[0360] Cy7-fluorescence recorded 24-hours after injecting tumor-bearing mice with platelets showed a structured Cy7 -fluorescence within the cranium of each of the three mice with tumors (Figures 28 A and 28B; Figure 23C), whereas very low Cy7-fluorescence signals were recorded in crania of mice in the control group (no intracranial tumor) (Figure 28C, 32A, and 32B). An MRI of the cranium of the mouse shown in Figure 28A recorded approximately 48-hours after injecting platelets confirmed colocalization of the Cy7/transferrin-platelets. The Tl -images of this mouse were recorded immediately before and immediately after injecting 100 lL of the contrast-enhancing MRI probe (Magnevist, 0.1M). Qualitative comparisons of the two images (Figure 28D (before contrast) and 28E (after contrast)) reveal a Magnevist-rich region in the cranium in the same vicinity as the Cy7-fluorescence that was recorded 24-hours earlier (Figure 28A).

[0361] Two sets of control studies were conducted to show the labeling of intra-cranial myeloma xenografts with KabC -platelets was dependent on interactions between transferrin molecules on the surface of platelets, and transferrin receptors on the surface of tumor cells.

[0362] In a first control study, mice without xenografts were injected with KabC-platelets coupled with Cy7 and transferrin. None of the mice from this control group (n=3) emitted a significant cranial fluorescence approximately 24-hours after platelet-injection. A representative Cy7-fluorescence image of a mouse from this group is shown in Figure 28C.

[0363] In a second control group, mice were injected intra-cranially with RFP-transfected U87 cells that over time developed neuroglioblastoma xenografts. These mice were injected with KabC-platelets coupled with Cy7 and transferrin. Neuroglioblastoma xenografts were resolved from images of RFP-fluorescence that were recorded 120-hours after injecting KabC-platelets (Figure 28F). The Cy7-fluorescence signal in the region of the RFP-labeled neuroglioblastoma xenografts was very weak (Figures 28G, 32D, and 32E). Transferrin receptor expression on U87 cells was analyzed by FACS of U87 cells incubated with a FITC-labeled anti-human transferrin receptor antibody. Only about one quarter of the U87 cells expressed transferrin receptor (Figure 28H).

[0364] These results demonstrate that transferrin-coupled KabC-platelets bind specifically to tumor cells that over-express the transferrin receptor, and moreover, transferrin-coupled KabC- platelets do not interact to a measurable degree with cells that lack or express low levels of the transferrin receptor, or non- specifically to other structures within the intra-cranial cavity. With the exception of the liver and spleen, which are responsible for platelet clearance, we did not observe a buildup of platelets in other tissues whose component cells express lower levels of the transferrin receptor compared to RPMI8226 cells and K562 cells. Moreover, the use of human transferrin on the platelet increased the specificity of binding to the human transferrin receptor molecules on the injected human tumor cells over murine transferrin receptor molecules expressed by the host mouse.

EXAMPLE 8: Immuno-histochemical analysis of platelets in myeloma xenografts [0365] Tissue slices prepared from xenografts that developed after intra-cranial injection of RPMI8226 cells in mice were analyzed by immuno-histocytochemistry. These studies demonstrate that KabC-platelets coupled with transferrin not only gain access to tumor microenvironments, but they accumulate there as a result of interactions between transferrin molecules on the platelets surface and receptor molecules on RPMI8226 cells.

[0366] KabC-platelets were loaded with chlorin e6, a membrane permeable Cy5-like fluorescence probe that can also serve as a photodynamic therapy probe. Chlorin e6 accumulates to a high level in the cytoplasm of KabC-platelets (Figure 23F). Chlorin e6-loaded KabC- platelets were divided into two fractions. The first fraction was surface-coupled with transferrin (test platelets). The second fraction lacked transferrin (control platelets). Control and test platelets were injected separately into the tail veins of mice (n=3) that had been injected intra- cranially 5 days earlier with RPMI8226 cells. Chlorin e6 fluorescence was recorded in live tumor-bearing mice using the Cy5 excitation and emission channel of the Caliper instrument.

[0367] The chlorin e6 fluorescence was weak in the crania of tumor-bearing mice injected with control platelets (Figure 29A, 32F, and 32G). In contrast, tumor-bearing mice injected with test KabC-platelets emitted a strong fluorescence from their crania (Figure 29B, 32H, and 321).

[0368] Tissue slices isolated from excised brains of mice injected with KabC-platelets loaded with chlorin e6 and surface-coupled with transferrin were fixed using 4% paraformaldehyde and frozen- sliced. The fixed tissue section was further labeled with a FITC-labeled antibody directed against the transferrin receptor. After extensive washing, regions from a representative slice of the multiple myeloma isolated from the mouse shown in Figure 29B were first observed visually, and imaged using bright-field (Figure 29D), and using the fluorescence emission from FITC (transferrin receptor; Figure 29C) and chlorin e6 (platelets; Figure 29E) channels of a Zeiss 700 confocal microscope. An overlay of the fluorescence images of FITC and chlorin e6 and the bright-field image is shown in Figure 29F.

[0369] FITC-fluorescence, which reflects the distribution of transferrin receptor on RPMI8826 cells, was not obvious in blood vessels although was uniformly distributed within tissue surrounding blood vessels (Figure 29C). Cross-sections of blood vessels in the tumor slice were labeled strongly with chlorin e6 (Figure 29E), consistent with the view that the majority of injected platelets are contained within blood vessels. The chlorin e6 fluorescence was strongest in the vicinity of blood vessels and decreased somewhat with increasing distance (Figure 29F). Chlorin e6 fluorescence within the tumor tissue was coincident in regions closer to blood vessels with cells that expressed the transferrin receptor (FITC-green), as can be seen from the pink- yellow colored regions in the overlap image. On the basis of analyses of results from the immunofluorescence-histocytochemical analysis and Cy7-fluorescence images of organs excised from tumor-bearing mice (Figure 27C), it can be concluded that KabC-platelets surface-coupled with transferrin are present within the tumor microenvironments and are retained through specific interactions with transferrin receptors on RPMI8826 cells.

Claims

CLAIMS What is claimed is:

1. A platelet composition comprising (i) an inactive platelet and (ii) a cargo, wherein the cargo is encapsulated within or attached to the surface of the inactivated platelet.

2. A platelet composition comprising (i) a platelet, (ii) a targeting group on the surface of the platelet, wherein the targeting group specifically binds to a target, and (iii) a cargo, wherein the cargo is encapsulated within or attached to the surface of the platelet.

3. A platelet composition comprising (i) a nano-platelet and (ii) a cargo, wherein the cargo is encapsulated within or attached to the surface of the nano-platelet.

4. The platelet composition according to claim 3, wherein the nano-platelet has a diameter of about 1000 nm or less.

5. The platelet composition according to claim 3 or 4, wherein the nano-platelet has a diameter between about 30 nm and about 500 nm.

6. The platelet composition according to any one of claims 3-5, wherein the nano-platelet has a diameter between about 150 nm and about 250 nm.

7. The platelet composition according to any one of claims 3-6, wherein the nano-platelet is derived from an activated platelet.

8. The platelet composition according to any one of claims 3-6, wherein the nano-platelet is derived from an inactive platelet.

9. The platelet composition according to any one of claims 3-8, wherein the nano-platelet is formed by sonicating, extruding, or enzymatic treatment of the platelet.

10. The platelet composition according to claim 2, wherein the platelet is an inactive platelet.

11. The platelet composition according to any one of claim 1 and 3-9, further comprising a targeting group on its surface, wherein the targeting group specifically binds to a target.

12. The platelet composition according to claim 2 or 11, wherein the target is a target cell, a target structure, or a soluble molecule.

13. The platelet composition according to any one of claims 2, 11, or 12, wherein the target is a cancer cell.

14. The platelet composition according to any one of claims 2, 11, or 12 wherein the target is an atherosclerotic plaque or an amyloid plaque.

15. The platelet composition according to claim 2 or 11-14, wherein the targeting group is transferrin, a polypeptide ligand, a small molecule ligand, an antibody, or an engineered scaffold that mimics an antibody.

16. The platelet composition according to any one of claims 2 and 11-15, wherein the targeting group is a polypeptide.

17. The platelet composition according to claim 16, wherein the polypeptide is expressed from a recombinant nucleic acid introduced into the platelet.

18. The platelet composition according to any one of claims 1-17, wherein the cargo comprises a therapeutic agent or a diagnostic agent.

19. The platelet composition according to any one of claims 1-18, wherein the cargo comprises a therapeutic agent and a diagnostic agent.

20. The platelet composition according to any one of claims 1-19 wherein the cargo comprises a magnetic nanoparticle.

21. The platelet composition according to any one of claims 1-20, wherein the cargo comprises a cytotoxic drug.

22. The platelet composition according to any one of claims 1-21, wherein the cargo comprises a fluorescent probe, a photodynamic therapy probe, a nanoparticle, a polymer, or an MRI contrast agent.

23. The platelet composition according to any one of claims 1-22, wherein the cargo comprises recombinant nucleic acid.

24. The platelet composition according to claim 23, wherein the recombinant nucleic acid encodes a therapeutic agent or a diagnostic agent.

25. The platelet composition according to claim 23 or 24, wherein the recombinant nucleic acid is encapsulated within the platelet or the fragment of the platelet.

26. The platelet composition according to claim 23 or 24, wherein the recombinant nucleic acid is coupled to the surface of the platelet or the fragment of the platelet.

27. The platelet composition according to any one of claims 1, 2 and 8-26, wherein the platelet is inactivated by contacting the platelet with an inactivating compound.

28. The platelet composition according to claim 27, wherein the inactivating compound is kabiramide C, aspirin, or prostaglandin E2.

29. The platelet composition according to any one of claims 17, 23, 25, and 26, wherein the recombinant nucleic acid is recombinant mRNA or recombinant cDNA

30. A composition comprising a polymer and the platelet composition according to any one of claims 1-29.

31. The composition according to claim 30, wherein the polymer is a hydrogel.

32. The composition according to claim 30 or 31, wherein the polymer is poly(N- isopropylacrylamide) .

33. A device for the manufacture of a platelet composition, the device comprising:

(i) a chamber;

(ii) a microfluidic conduit;

(iii) one or more reagent ports fluidly connected to the microfluidic conduit; and

(iv) a concentrator, wherein the microfluidic conduit connects the chamber and the concentrator.

34. The device according to claim 33, wherein the device further comprises a sonicator.

35. The device according to claim 33 or 34, wherein the one or more reagent ports provides an inactivating compound, a targeting group, a diagnostic agent, and/or a therapeutic agent.

36. The device according to any one of claims 33-35, wherein the chamber is a culture chamber.

37. The device according to claim 36, wherein the culture chamber comprises a matrix that supports cellular growth.

38. The device according to claim 37, wherein the matrix that supports cellular growth comprises a hydrogel.

39. A method of forming the platelet composition according to any one of claims 1 and 11- 29 comprising:

(i) contacting a platelet with an inactivating compound; and

(ii) loading the inactive platelet with the cargo or attaching the cargo to the surface of the inactive platelet.

40. A method of forming the platelet composition according to any one of claims 1 and 11- 29 comprising:

(i) loading a platelet with the cargo or attaching the cargo to the surface of a platelet; and

(ii) contacting the platelet with an inactivating compound.

41. A method of forming the platelet composition according to any one of claims 2 and 11- 29 comprising:

(i) attaching the targeting group to a surface of a platelet; and

(ii) loading the platelet with the cargo or attaching the cargo to the surface of a platelet.

42. A method forming the platelet composition according to any one of claims 3-9 and 11-29 comprising:

(i) fragmenting a platelet to form a nano-platelet; and

(ii) loading the nano-platelet with the cargo or attaching the cargo to the surface of a platelet.

43. A method forming the platelet composition according to any one of claims 3-9 and 11-29 comprising:

(ii) fragmenting the platelet to form nano-platelets.

44. The method according to any one of claims 39-43, further comprising culturing a cytomegakaryocyte to produce the platelet.

45. The method according to any one of claims 44, further comprising transfecting the cytomegakaryocte with a recombinant nucleic acid.

46. The method according to claim 45, wherein the recombinant nucleic acid is recombinant mRNA or recombinant cDNA.

47. The method according to claim 45 or 46, wherein the recombinant nucleic acid encodes a targeting group, a therapeutic agent, or a diagnostic agent.

48. A method of treating a disease in an individual comprising administering to the individual the platelet composition according to any one of claims 1-29.

49. A method of treating a disease in an individual comprising administering to the individual the composition according to any one of claims 30-32.

50. A method of diagnosing a disease in an individual comprising administering to the individual the platelet composition according to any one of claims 1-29.

51. A method of diagnosing and treating a disease in an individual comprising administering to the individual the platelet composition according to any one of claims 1-29.

52. The method of diagnosing and treating a disease in an individual according to claim 51, wherein the platelet composition comprises a diagnostic agent and a therapeutic agent.

53. The method according to any one of claims 48-52, wherein the disease is cancer, Alzheimer's disease, or atherosclerosis.

54. A method of producing the platelet composition according to any one of claims 1-29 using the device according to any one of claims 33-38 comprising:

(i) contacting a platelet with an inactivating compound; and

55. The method according to claim 54, further comprising culturing cytomegakaryocytes in the chamber to produce platelets.

56. The method according to claim 54 or 55, further comprising attaching a targeting group to the surface of the platelet, wherein the targeting group specifically binds to a target.

57. The method according to any one of claims 54-56, further comprising fragmenting the platelets.

58. The method according to any one of claims 54-57, further comprising sonicating the platelets.

59. The platelet composition according to claim 13, wherein the cancer cell is a multiple myeloma cell or a leukemia cell.

60. The platelet composition according to claim 27, wherein the inactivating compound is salicylic acid.

61. The platelet composition according to claim 27, wherein the inactivating compound is cytochalasin.