US 20200206143A1 IN (19 ) United States ( 12) Patent Application Publication (10 ) Pub . No.: US 2020/0206143 A1 Moskowitz et al. (43 ) Pub . Date : Jul. 2 , 2020

( 54 ) PLATELETS LOADED WITH ANTI - CANCER Publication Classification AGENTS (51 ) Int. Ci. A61K 9/50 (2006.01 ) ( 71 ) Applicant: Cellphire, Inc., Rockville , MD (US ) A61K 31/704 (2006.01 ) ( 72 ) Inventors : Keith Andrew Moskowitz , Westfield , A61K 31/502 ( 2006.01 ) IN (US ) ; Rafael Jorda, Bethesda, MD A61K 31/337 (2006.01 ) (US ) ; Ying Yi Zheng , Rockville , MD AOIN 1/02 ( 2006.01) (US ) ; Daniel Sheik , Rockville , MD A61K 9/127 (2006.01 ) (US ) (52 ) U.S. CI. CPC A61K 9/5068 ( 2013.01 ) ; A61K 31/704 (2013.01 ) ; A61K 31/502 ( 2013.01) ; AOIN ( 21 ) Appl. No .: 16 /698,645 1/021 ( 2013.01) ; AOIN 1/0226 (2013.01 ) ; A61K 9/1271 (2013.01 ) ; A61K 31/337 ( 22 ) Filed : Nov. 27 , 2019 (2013.01 ) ( 57 ) ABSTRACT Related U.S. Application Data In some embodiments provided herein is a method of (60 ) Provisional application No.62 / 773,931, filed on Nov. preparing cargo - loaded platelets , comprising : treating plate 30, 2018 , provisional application No. 62/ 775,141 , lets with a cargo and with a loading buffer comprising a salt , filed on Dec. 4 , 2018, provisional application No. a base , a loading agent , and optionally ethanol, to form the 62 /828,041 , filed on Apr. 2 , 2019 . cargo - loaded platelets . Patent Application Publication Jul. 2 , 2020 Sheet 1 of 20 US 2020/0206143 A1

Amount of (DOX ) load per CD42b * Platelet

DOX(mfi)

FIG . 1

Amount of DOX in drug loaded platelets following ADP and /or TRAP stimulation

227

FIG . 2 Patent Application Publication Jul. 2 , 2020 Sheet 2 of 20 US 2020/0206143 A1

Loading platelets with DOX encapsulated liposome

DOXmfi()

0 Cells 250ul 100ml Lipo - DOX

FIG . 3 Patent Application Publication Jul. 2 , 2020 Sheet 3 of 20 US 2020/0206143 A1

2h Incubation

Control 0.1mM DOX W20.3mM DOX

0 104 106 Control 95.1 % DOX loaded

B

Doxorubicin

FIG . 4A - C Patent Application Publication Jul. 2 , 2020 Sheet 4 of 20 US 2020/0206143 A1

Control 0.5h incubation

92.7 % 2.OK DOX loaded 1.5K

CellCount 102 106 Doxorubicin

FIG . 5 Patent Application Publication Jul. 2 , 2020 Sheet 5 of 20 US 2020/0206143 A1

15 Doxosome ( umol) FIGS. 6A (left ) & 6B (right )

25000 y 996.6x - 344.92 20000 R2 a 0.9978

Fluorescence 10000

0 0 Doxorubicin (UM )

FIG.7 Patent Application Publication Jul. 2 , 2020 Sheet 6 of 20 US 2020/0206143 A1

DOX Quantification

2 3 Membrane bound 108K 235K 454K 915K 1448K 1745K Cells / ul

FIG . 8

DOX(mg)/1platelet 2 26-06

108K 235K 454K 915K 1448K 1745K CellsiuL

FIG.9 Patent Application Publication Jul. 2 , 2020 Sheet 7 of 20 US 2020/0206143 A1

2

5

FIG . 10 Patent Application Publication Jul. 2 , 2020 Sheet 8 of 20 US 2020/0206143 A1

N

FIG . 11 Patent Application Publication Jul. 2 , 2020 Sheet 9 of 20 US 2020/0206143 A1

Loading buffer allowed higher drug load per cell than compared to HMTbuffer

52A

FIG . 12

DOX Induces Aggregation of Platelets 100 96 90 85 . 80 70 60 50 40 TransmittanceLight% 30 de 20 14 . 10 0 HMT + 1mM Thrombin 0.36mg/ ml 0.72mg/ ml Img/ ml DOX 3mg/ ml DOX Mg liliDOX DOX FIG . 13A Patent Application Publication Jul. 2 , 2020 Sheet 10 of 20 US 2020/0206143 A1

FIG . 13B

FIG . 13C Patent Application Publication Jul. 2 , 2020 Sheet 11 of 20 US 2020/0206143 A1

Gate : Singlet platelets 3 PARP -FL No drug 4.72 % 10?M PARP?- FL , 0.5MM PARPI count(x103/UL)Cell loaded 50.43 % 30uM PARPL- FL , 1.5MM PARPI 1.81.2 platelets 97.11 % 60LM PARP - FL , 3mM PARPI 99.97 % 120 M PARPL- FL , 6MM PARPI 0.6 Method : flow cytometry - 3. pooled random donor platelets 0 Post 3 hour incubation at 37 ° C 101 102 103 104 105 106 107.2 Patent Application Publication Jul. 2 , 2020 Sheet 12 of 20 US 2020/0206143 A1

Total drug load / cell

* Method Tecan fluorometer 2.0E - 09 Exem , 450 /518nm

)(mg/call (PARP?-FL+PARPI)/platelet pooled random donor platelets

Ttest, 2 tails * p < 0.05 p < 0.01

PARPI- FL (UM ) 0 30 120 PARPI (MM ) 0 0.5 3.0 6.0 FIG . 15

FIG . 16 Patent Application Publication Jul. 2 , 2020 Sheet 13 of 20 US 2020/0206143 A1

FIG . 17

FIG . 18 Patent Application Publication Jul. 2 , 2020 Sheet 14 of 20 US 2020/0206143 A1

FIG . 19

FIG . 20 Patent Application Publication Jul . 2 , 2020 Sheet 15 of 20 US 2020/0206143 A1

.

FIG . 21

FG. 22 Patent Application Publication Jul. 2 , 2020 Sheet 16 of 20 US 2020/0206143 A1

Unloaded Tsomes DOX loaded Tsomes DOX loaded Tsomes Cephalin 4 ThrombinPeakHeight Octaplas 5

20

0 everythin 30.67 36.67 42.67 72.67 Time (min )

FIG . 23 Patent Application Publication Jul. 2 , 2020 Sheet 17 of 20 US 2020/0206143 A1

HSA medium (neg control) 1 % Dox loaded 1 % unloaded 5 % Dox loaded muy 5 % unloaded 10 % Dox loaded Dox (positive control ) Populationdoublings

Time after adding fresh medium (hrs )

FIG . 24 Patent Application Publication Jul. 2 , 2020 Sheet 18 of 20 US 2020/0206143 A1

E 30003

thanMore 0 2

FIG . 25A

0 4 6 (hours )

FIG . 25B Patent Application Publication Jul. 2 , 2020 Sheet 19 of 20 US 2020/0206143 A1 1000KM

1 FIG . 26A

1

100

0 3 2 (hours ) FIG . 260 Patent Application Publication Jul. 2 , 2020 Sheet 20 of 20 US 2020/0206143 A1

Brightfield Fluorescence Overlay

FIG . 27 US 2020/0206143 A1 Jul . 2 , 2020 1

PLATELETS LOADED WITH ANTI - CANCER used to control bleeding after injury or during acquired AGENTS platelet function defects or deficiencies, for example those occurring during surgery and those due to the presence of CROSS - REFERENCE TO RELATED platelet inhibitors . APPLICATIONS [0006 ] Loading platelets with pharmaceutical drugs may allow targeted delivery of the drugs to sites of interest . [0001 ] This application claimspriority to U.S. Provisional Further, drug - loaded platelets may be lyophilized or cryo Patent Application No. 62/ 773,931 , filed on Nov. 30 , 2018 , preserved to allow for long - term storage . In some embodi U.S. Provisional Patent Application No. 62/ 775,141 , filed on ments the loading of a drug in the platelets mitigates Dec. 4 , 2018 , and U.S. Provisional Patent Application No. systemic side effects associated with the drug and lowers the 62/ 828,041 , filed on Apr. 2 , 2019. The contents of each of threshold of therapeutic dose necessary by facilitating tar these applications are incorporated herein by reference in geted treatment at site of interest . See , Xu P. , et. al. , their entireties. Doxorubicin - loaded platelets as a smart drug delivery sys tem : An improved therapy for lymphoma, Scientific Reports , TECHNICAL FIELD 7 , Article Number: 42632 , (2017 ), which is incorporated by [ 0002 ] The present disclosure in some embodiments reference herein in its entirety . relates to the use of platelets , platelet derivatives , or throm bosomes ( e.g., freeze -dried platelet derivatives ) as biologi SUMMARY OF THE INVENTION cal carriers of cargo , such as pharmaceutical drugs, also [0007 ] In some embodiments provided herein is a method referred to herein as drug - loaded platelets , platelet deriva of preparing cargo - loaded platelets , cargo - loaded platelet tives , or thrombosomes . Also provided herein in some derivatives , or cargo -loaded thrombosomes (e.g. , freeze embodiments are methods of preparing platelets , platelet dried platelet derivatives ) , comprising : derivatives, or thrombosomes loaded with the drug of inter [0008 ] treating platelets , platelet derivatives, or throm est . bosomes with a cargo and with at least one loading [0003 ] The present disclosure relates to the field of blood agent and optionally one or more plasticizers such as and blood products . More specifically , it relates to platelets , organic solvents , such as organic solvents selected from cryopreserved platelets , and /or lyopreserved platelet com the group consisting of ethanol, acetic acid , acetone, positions, including those containing stabilized platelets or acetonitrile , dimethylformamide , dimethyl sulfoxide, compositions derived from platelets . The drug - loaded plate dioxane , methanol, n - propanol , isopropanol , tetrahy lets can be stored under typical ambient conditions, refrig drofuran ( THF ), N -methyl pyrrolidone , dimethylacet erated , cryopreserved , for example with dimethyl sulfoxide amide (DMAC ), or combinations thereof, (DMSO ), and /or lyophilized after stabilization ( e.g. , throm to form the cargo - loaded platelets , cargo - loaded platelet bosomes ) derivatives, or cargo - loaded thrombosomes. [0009 ] In some embodiments , the method of preparing BACKGROUND cargo - loaded platelets can include treating the platelets , the [0004 ] Blood is a complex mixture of numerous compo platelet derivatives, and/ or the thrombosomes with the cargo nents . In general , blood can be described as comprising four with one loading agent. In some embodiments , the method main parts : red blood cells , white blood cells , platelets , and of preparing cargo - loaded platelets , cargo - loaded platelet plasma. The first three are cellular or cell- like components , derivatives , or cargo - loaded thrombosomes can include whereas the fourth ( plasma ) is a liquid component compris treating the platelets , the platelet derivatives , or the throm ing a wide and variable mixture of salts , proteins, and other bosomes with the cargo with multiple loading agents . factors necessary for numerous bodily functions. The com [0010 ] In some embodiments , suitable organic solvents ponents of blood can be separated from each other by include , but are not limited to alcohols, esters, ketones , various methods. In general, differential centrifugation is ethers , halogenated solvents , hydrocarbons, nitriles , glycols , most commonly used currently to separate the different alkyl nitrates, water or mixtures thereof. In some embodi components of blood based on size and , in some applica ments , suitable organic solvents includes , but are not limited tions , density . to methanol, ethanol, n -propanol , isopropanol, acetic acid , [ 0005 ] Unactivated platelets , which are also commonly acetone, methyl ethyl ketone, methyl isobutyl ketone , referred to as thrombocytes, are small, often irregularly methyl acetate , ethyl acetate , isopropyl acetate , tetrahydro shaped ( e.g., discoidal or ovoidal ) megakaryocyte -derived furan , isopropyl ether ( IPE ), tert- butyl methyl ether, dioxane components of blood that are involved in the clotting ( e.g. , 1,4 -dioxane ), acetonitrile , propionitrile , methylene process . They aid in protecting the body from excessive chloride, chloroform , toluene , anisole , cyclohexane , hexane , blood loss due not only to trauma or injury , but to normal heptane, ethylene glycol, nitromethane , dimethylforma physiological activity as well. Platelets are considered cru mide , dimethyl sulfoxide, N -methyl pyrrolidone , dimethyl cial in normal hemostasis , providing the first line of defense acetamide, and combinations thereof. The presence of against blood escaping from injured blood vessels . Platelets organic solvents , such as ethanol, can be beneficial in the generally function by adhering to the lining ofbroken blood processing of platelets , platelet derivatives , and /or throm vessels , in the process becoming activated , changing to an bosomes. In particular , the organic solvent may open up amorphous shape , and interacting with components of the and /or increase the flexibility of the plasma membrane of the clotting system that are present in plasma or are released by platelets , platelet derivatives, and /or thrombosomes, which the platelets themselves or other components of the blood . allows a higher amount of cargo ( e.g., drug ) to be loaded into Purified platelets have found use in treating subjects with the platelets, platelet derivatives, and /or thrombosomes. In low platelet count ( thrombocytopenia ) and abnormal platelet some embodiments , the organic solvent can aid in solubi function ( thrombasthenia ). Concentrated platelets are often lizing molecules to be loaded . US 2020/0206143 A1 Jul. 2. 2020 2

[0011 ] In some embodiments provided herein is a method platelet derivatives, or the drug - loaded thrombosomes. In of preparing drug- loaded platelets , drug -loaded platelet some embodiments , the method further comprises cold derivatives, or drug -loaded thrombosomes , comprising : storing the drug - loaded platelets , drug - loaded platelet [0012 ] treating platelets , platelet derivatives, or throm derivatives , or the drug - loaded thrombosomes. In some bosomes with a drug and with a loading buffer com embodiments , the method further comprises drying the prising a base , a loading agent, and optionally at least drug - loaded platelets or the drug - loaded platelet derivatives . one organic solvent such as an organic solvent selected In some embodiments , the method further comprises freeze from the group consisting of ethanol, acetic acid , drying the drug - loaded platelets or the drug - loaded platelet acetone, acetonitrile , dimethylformamide , dimethyl derivatives . In such embodiments , the method may further sulfoxide, dioxane ,methanol , n - propanol, isopropanol, comprise rehydrating the drug - loaded platelets, drug - loaded tetrahydrofuran ( THF ), N -methyl pyrrolidone , dim platelet derivatives, or drug - loaded thrombosomes obtained ethylacetamide (DMAC ), or combinations thereof, from the drying step . to form the drug -loaded platelets , the drug -loaded platelet [0027 ] In some embodiments , the method that further derivatives, or the drug - loaded thrombosomes . comprises drying the drug - loaded platelets or drug - loaded [0013 ] In some embodiments provided herein is a method platelet derivatives and rehydrating the drug - loaded platelets of preparing cargo - loaded platelets , cargo -loaded platelet or the drug - loaded platelet derivatives obtained from the derivatives, or cargo - loaded thrombosomes, comprising: drying step provides rehydrated platelets or platelet deriva [0014 ) treating platelets , platelet derivatives , or throm tives comprising at least 10 % of the amount of the drug of bosomes with a cargo and with a loading buffer com step (b ). prising a salt , a base , a loading agent, and optionally at [0028 ] In some embodiments, the method that further least one organic solvent comprises drying the drug - loaded platelets or the drug to form the cargo - loaded platelets , cargo - loaded platelet loaded platelet derivatives and rehydrating the drug -loaded derivatives, or the cargo - loaded thrombosomes. platelets or the drug - loaded platelet derivatives obtained [0015 ] In some embodiments provided herein is a method from the drying step provides rehydrated platelets or platelet of preparing cargo - loaded platelets , cargo - loaded platelet derivatives , or cargo -loaded thrombosomes, comprising : derivatives comprising from about 1 nM to about 100 mm , [ 0016 ] treating platelets , platelet derivatives , or throm such as about 10 nM to about 10 mM , such as about 100 nM bosomes with a cargo and with a loading agent and to 1 mM , of the drug . optionally at least one organic solvent [ 0029 ] In some embodiments , the platelets , platelet to form the cargo -loaded platelets , the cargo - loaded platelet derivatives, or thrombosomes are treated with the drug and derivatives, or the cargo - loaded thrombosomes . with the buffer sequentially, in either order . [0017 ] In some embodiments provided herein is a method [0030 ] Thus, in some embodiments provided herein is a of preparing drug - loaded platelets , drug - loaded platelet method of preparing drug -loaded platelets , drug -loaded derivatives , or drug- loaded thrombosomes, comprising: platelet derivatives , or drug - loaded thrombosomes, compris [0018 ] treating platelets , platelet derivatives, or throm ing : bosomes with a drug and with a loading buffer com [0031 ] (1 ) treating platelets , platelet derivatives, or prising a base , a loading agent, and optionally at least thrombosomes with a drug to form a first composition ; one organic solvent and to form the drug -loaded platelets , the drug- loaded platelet (0032 ] (2 ) treating the first composition with a buffer derivatives, or the drug -loaded thrombosomes. comprising a salt , a base, a loading agent, and option [0019 ] In some embodiments provided herein is a method ally at least one organic solvent to form the drug - loaded of preparing drug- loaded platelets , drug -loaded platelet platelets , drug - loaded platelet derivatives, or drug derivatives , or drug - loaded thrombosomes , comprising : loaded thrombosomes . [0020 ] treating platelets , platelet derivatives , or throm [0033 ] In some embodiments of the methods of preparing bosomes with a drug and with a loading buffer com cargo - loaded platelets, such as drug - loaded platelets , as prising a salt , a base, a loading agent, and optionally at provided herein , the methods do not comprise treating least one organic solvent platelets , platelet derivatives , or thrombosomes with etha to form the drug - loaded platelets , the drug -loaded platelet nol. derivatives, or the drug - loaded thrombosomes. [0034 ] In some embodiments of the methods of preparing [ 0021 ] In some embodiments provided herein is a method cargo - loaded platelets , such as drug - loaded platelets , as of preparing drug - loaded platelets , drug - loaded platelet provided herein , the methods do not comprise treating derivatives, or drug - loaded thrombosomes, comprising: platelets , platelet derivatives, or thrombosomes with a sol [ 0022 ] a ) providing platelets , platelet derivatives, or vent selected from the group consisting of ethanol, acetic thrombosomes; acid , acetone, acetonitrile, dimethylformamide, dimethyl [0023 ] and sulfoxide , dioxane , methanol, n -propanol , isopropanol, tet [0024 ] b ) treating the platelets , the platelet derivatives , rahydrofuran ( THF ), N -methyl pyrrolidone , dimethylacet or the thrombosomes with a drug and with a loading amide (DMAC ) , or combinations thereof. buffer comprising a salt , a base , a loading agent, and [0035 ] In some embodiments of the methods of preparing optionally at least one organic solvent cargo - loaded platelets , such as drug- loaded platelets , as [0025 ] to form the drug - loaded platelets , drug - loaded provided herein , the methods do not comprise treating platelet derivatives , or the drug - loaded thrombo platelets , platelet derivatives, or thrombosomes with an somes . organic solvent. [0026 ] In some embodiments , the method further com [0036 ] In some embodiments of the methods of preparing prises cryopreserving the drug -loaded platelets , drug -loaded cargo - loaded platelets , such as drug - loaded platelets , as US 2020/0206143 A1 Jul . 2 , 2020 3

provided herein , the methods do not comprise treating the drug - loaded platelet derivatives. In such embodiments , platelets , platelet derivatives, or thrombosomes with a sol the method may further comprise rehydrating the drug vent. loaded platelets or the drug - loaded platelet derivatives [0037 ] In some embodiments of the methods of preparing obtained from the drying step . cargo - loaded platelets , such as drug - loaded platelets , as [0045 ] In some embodiments , the method that further provided herein , the methods comprise treating platelets , comprises drying the drug - loaded platelets or the drug platelet derivatives , or thrombosomes with a solvent, such as loaded platelet derivatives and rehydrating the drug -loaded an organic solvent, such as organic solvent selected from the platelets or the drug -loaded platelet derivatives obtained group consisting of ethanol, acetic acid , acetone , acetoni from the drying step provides rehydrated platelets or platelet trile , dimethylformamide, dimethyl sulfoxide , dioxane , derivatives comprising at least 10 % of the amount of the methanol, n - propanol, isopropanol, tetrahydrofuran ( THF) , drug of step ( 2 ) N -methylpyrrolidone , dimethylacetamide (DMAC ), or [ 0046 ] In some embodiments , the method that further combinations thereof, such as ethanol. comprises drying the drug - loaded platelets or the drug [0038 ] In some embodiments , the method further com loaded platelet derivatives and rehydrating the drug - loaded prises drying the drug -loaded platelets or the drug -loaded platelets or the drug - loaded platelet derivatives obtained platelet derivatives obtained in step ( 2 ). In some embodi from the drying step provides rehydrated platelets or throm ments , the method further comprises cryopreserving , lyo bosomes comprising from about 1 nM to about 1000 mm , preserving ( e.g., freeze -drying ) the drug - loaded platelets or such as about 10 nM to about 10 mM , such as about 100 nM the drug - loaded platelet derivatives. In some embodiments , to 1 mM , of the drug of step ( 2 ) . the method further comprises cold storing the drug- loaded [0047 ] In some embodiments , the platelets or thrombo platelets , the drug - loaded platelet derivatives, the drug somes are treated with the drug and with the buffer concur loaded thrombosomes , or compositions containing drug rently loaded platelets at suitable storage temperatures, such as [0048 ] Thus , in some embodiments provided herein is a standard room temperature storing ( e.g., storing at a tem method of preparing drug - loaded platelets , the drug - loaded perature ranging from about 20 to about 30 ° C.) or cold platelet derivatives, or the drug - loaded thrombosomes, com storing ( e.g., storing at a temperature ranging from about 1 prising: to about 10 ° C.) . In some embodiments , the method further [0049 ] treating the platelets , the platelet derivatives, or comprises cryopreserving , freeze - drying, thawing, rehydrat the thrombosomes with a drug in the presence of a ing , and combinations thereof, the drug loaded platelets , the buffer comprising a salt, a base , a loading agent , and drug - loaded platelet derivatives , or the drug - loaded throm optionally ethanol, to form the drug - loaded platelets , bosomes. For example , in such embodiments , the method the drug - loaded platelet derivatives, or the drug - loaded may further comprise rehydrating the drug - loaded platelets , thrombosomes . the drug - loaded platelet derivatives, or the drug - loaded [0050 ] In some embodiments , the method further com thrombosomes obtained from the drying step . prises drying the drug - loaded platelets or the drug - loaded [0039 ] In some embodiments , the method that further platelet derivatives . In some embodiments , the method fur comprises drying the drug -loaded platelets or drug - loaded ther comprises freeze -drying the drug -loaded platelets or the platelet derivatives and rehydrating the drug- loaded platelets drug - loaded platelet derivatives . In such embodiments , the or the drug - loaded platelet derivatives obtained from the method may further comprise rehydrating the drug - loaded drying step provides rehydrated platelets or platelet deriva platelets or the drug - loaded platelet derivatives obtained tives comprising at least 10 % of the amount of the drug of from the drying step . step ( 1 ) [0051 ] In some embodiments , the method that further [0040 ] In some embodiments , the method that further comprises drying the drug - loaded platelets or the drug comprises drying the drug -loaded platelets or the drug loaded platelet derivatives and rehydrating the drug -loaded loaded platelet derivatives and rehydrating the drug - loaded platelets or the drug - loaded platelet derivatives obtained platelets or the drug - loaded platelet derivatives obtained from the drying step provides rehydrated platelets or the from the drying step provides rehydrated platelets or platelet thrombosomes comprising at least 10 % of the amount of the derivatives comprising from about 1 nM to about 1000 mm , drug prior to loading . such as about 10 nM to about 100 mM , such as about 100 [ 0052 ] In some embodiments , the method that further nM to 10 mM , of the drug of step ( 1) . comprises drying the drug - loaded platelets or the drug [0041 ] In some embodiments provided herein is a method loaded platelet derivatives and rehydrating the drug -loaded of preparing drug - loaded platelets , drug - loaded platelet platelets or the drug - loaded platelet derivatives obtained derivatives , or drug- loaded thrombosomes , comprising : from the drying step provides rehydrated platelets or throm [0042 ] ( 1) treating the platelets , platelet derivatives , or bosomes comprising from about 1 nM to about 1000 mM , thrombosomes with a buffer comprising a salt, a base , such as about 10 nM to about 10 mM , such as about 100 nM a loading agent, and optionally ethanol, to form a first to 1 mM , of the drug . composition ; and [0053 ] In some embodiments , platelets , platelet deriva [0043 ] ( 2 ) treating the first composition with a drug , to tives , or thrombosomes are pooled from a plurality of form the drug - loaded platelets , the drug - loaded platelet donors . Such platelets , platelet derivatives , and thrombo derivatives , or the drug - loaded thrombosomes. somes pooled from a plurality of donors may be also referred [0044 ] In some embodiments, the method further com herein to as pooled platelets , platelet derivatives, or throm prises drying the drug - loaded platelets , the drug - loaded bosomes . In some embodiments , the donors are more than 5 , platelet derivatives, or the drug- loaded thrombosomes such as more than 10 , such as more than 20 , such as more obtained in step ( 2 ) . In some embodiments , the method than 50 , such as up to about 100 donors . In some embodi further comprises freeze - drying the drug -loaded platelets or ments , the donors are from about 5 to about 100 , such as US 2020/0206143 A1 Jul . 2 , 2020 4

from about 10 to about 50 , such as from about 20 to about loaded platelet derivatives and rehydrating the drug -loaded 40 , such as from about 25 to about 35 . platelets or the drug - loaded platelet derivatives obtained [0054 ] Thus, provided herein in some embodiments is a from the drying step provides rehydrated platelets or rehy method of preparing drug - loaded platelets , drug- loaded drated platelet derivatives comprising at least 10 % of the platelet derivatives , or drug - loaded thrombosomes compris amount of the drug of step ( B ) ( 1 ) . ing [ 0067] In some embodiments , the method that further [0055 ] A ) pooling platelets , platelet derivatives, or comprises drying the drug - loaded platelets or the drug thrombosomes from a plurality of donors ; and loaded platelet derivatives and rehydrating the drug -loaded [0056 ] B ) treating the platelets , platelet derivatives, or platelets or the drug - loaded platelet derivatives obtained thrombosomes from step ( A ) with a drug and with a from the drying step provides rehydrated platelets or platelet loading buffer comprising a salt, a base , a loading derivatives comprising from about 1 nM to about 1000 mm , agent , and optionally ethanol , to form the drug - loaded such as about 10 nM to about 10 mM , such as about 100 nM platelets , the drug - loaded platelet derivatives , or the to 1 mM , of the drug of step ( B ) (1 ). drug -loaded thrombosomes . [0068 ] Thus , provided herein in some embodiments is a [0057 ] In some embodiments , the method further com method of preparing drug - loaded platelets , drug - loaded prises drying the drug - loaded platelets or the drug - loaded platelet derivatives, or drug - loaded thrombosomes compris platelet derivatives obtained in step ( B ) . In some embodi ing : ments , the method further comprises freeze -drying the drug loaded platelets or the drug - loaded platelet derivatives . In [0069 ] A ) pooling platelets , platelet derivatives , or such embodiments , the method may further comprise rehy thrombosomes from a plurality of donors ; and B ) drating the drug - loaded platelets or the drug - loaded platelet [0070 ] ( 1) treating the platelets , the platelet deriva derivatives obtained from the drying step . tives , or the thrombosomes from step ( A ) with a [0058 ] In some embodiments , the method that further buffer comprising a salt, a base , a loading agent, and comprises drying the drug - loaded platelets or the drug optionally ethanol, to form a first composition ; and loaded platelet derivatives and rehydrating the drug - loaded [0071 ] (2 ) treating the first composition with a drug platelets or the drug - loaded platelet derivatives obtained to form the drug - loaded platelets , the drug - loaded from the drying step provides rehydrated platelets or rehy platelet derivatives, or the drug -loaded thrombo drated platelet derivatives comprising at least 10 % of the somes . amount of the drug of step ( B ) . [ 0072 ] In some embodiments , the method further com [0059 ] In some embodiments , the method that further prises drying the drug - loaded platelets or the drug - loaded comprises drying the drug - loaded platelets or the drug platelet derivatives obtained in step ( B ) (2 ) . In some embodi loaded platelet derivatives and rehydrating the drug - loaded ments , the method further comprises freeze - drying the drug platelets or the drug - loaded platelet derivatives obtained loaded platelets or the drug -loaded platelet derivatives. In from the drying step provides rehydrated platelets or rehy such embodiments , the method may further comprise rehy drated platelet derivatives comprising from about 1 nM to drating the drug - loaded platelets or the drug - loaded platelet about 1000 mM , such as about 10 nM to about 10 mM , such derivatives obtained from the drying step . as about 100 nM to 1 mM , of the drug of step ( B ) . [ 0073 ] In some embodiments , the method that further [ 0060 ] In some embodiments, the pooled platelets , platelet comprises drying the drug -loaded platelets or the drug derivatives , or thrombosomes are treated with the drug and loaded platelet derivatives and rehydrating the drug -loaded with the buffer sequentially , in either order . platelets or the drug - loaded platelet derivatives obtained [0061 ] Thus, provided herein in some embodiments is a from the drying step provides rehydrated platelets or throm method of preparing drug - loaded platelets , drug - loaded bosomes comprising at least 10 % of the amount of the drug platelet derivatives , or drug -loaded thrombosomes compris of step (B )( 2 ). ing : [0074 ] In some embodiments , the method that further [0062 ] A ) pooling platelets , platelet derivatives , or comprises drying the drug - loaded platelets or the drug thrombosomes from a plurality of donors ; and B ) loaded platelet derivatives and rehydrating the drug -loaded [ 0063 ] ( 1 ) treating the platelets , platelet derivatives , platelets or the drug - loaded platelet derivatives obtained or thrombosomes from step ( A ) with a drug to form from the drying step provides rehydrated platelets or throm a first composition ; and bosomes comprising from about 1 nM to about 1000 mm , [0064 ] (2 ) treating the first composition with a buffer such as about 10 nM to about 10 mM , such as about 100 nM comprising a salt, a base , a loading agent, and to 1 mM , of the drug of step (B ) (2 ). optionally ethanol, to form the drug - loaded platelets , [0075 ] In some embodiments , the pooled platelets , platelet drug -loaded platelet derivatives, or drug - loaded derivatives, or thrombosomes are treated with the drug and thrombosomes . with the buffer concurrently . [0065 ] In some embodiments , the method further com prises drying the drug - loaded platelets or the drug - loaded [0076 ] Thus , in some embodiments provided herein is a platelet derivatives obtained in step ( B ) ( 2 ) . In some embodi method of preparing drug -loaded platelets , drug -loaded ments , the method further comprises freeze - drying the drug platelet derivatives , or drug - loaded thrombosomes , compris loaded platelets or the drug -loaded platelet derivatives . In ing : such embodiments , the method may further comprise rehy [0077 ] A ) pooling platelets , platelet derivatives, or drating the drug - loaded platelets or the drug - loaded platelet thrombosomes from a plurality of donors ; and derivatives obtained from the drying step . [0078 ] B ) treating the platelets , the platelet derivatives , [ 0066] In some embodiments , the method that further or the thrombosomes with a drug in the presence of a comprises drying the drug - loaded platelets or the drug buffer comprising a salt, a base , a loading agent, and US 2020/0206143 A1 Jul. 2. 2020 5

optionally ethanol , to form the drug - loaded platelets , as EGTA . In some embodiments provided herein are drug the drug - loaded platelet derivatives , or the drug - loaded loaded platelets, drug - loaded platelet derivatives , or drug thrombosomes . loaded thrombosomes that are not prepared with a chelating [0079 ] In some embodiments , the method further com agent such as EGTA . prises drying the drug -loaded platelets or the drug -loaded [0088 ] In some embodiments provided herein the method platelet derivatives obtained in step ( B ). In some embodi includes treating the first composition with Prostaglandin 1 ments , the method further comprises freeze- drying the drug (PGE1 ) or Prostacyclin . In some embodiments provided loaded platelets or the drug - loaded platelet derivatives. In herein the method does not include treating the first com such embodiments , the method may further comprise rehy position with Prostaglandin 1 ( PGE1) or Prostacyclin . drating the drug - loaded platelets or the drug - loaded platelet [0089 ] In some embodiments provided herein the method derivatives obtained from the drying step . includes treating the first composition with a chelating agent [0080 ] In some embodiments , the method that further such as EGTA . In some embodiments provided herein the comprises drying the drug -loaded platelets or the drug method does not include treating the first composition with loaded platelet derivatives and rehydrating the drug - loaded a chelating agent such as EGTA . platelets or the drug - loaded platelet derivatives obtained [0090 ] In some embodiments provided herein are drug from the drying step provides rehydrated platelets or throm loaded platelets , drug -loaded platelet derivatives, or drug bosomes comprising at least 10 % of the amount of the drug loaded thrombosomes prepared with an anti- aggregation of step ( B ). agent. [ 0081] In some embodiments , the method that further [0091 ] In some embodiments provided herein are drug comprises drying the drug - loaded platelets or the drug loaded platelets , drug -loaded platelet derivatives, or drug loaded platelet derivatives and rehydrating the drug - loaded loaded thrombosomes prepared with an anti- aggregation platelets or the drug - loaded platelet derivatives obtained agent such as GPIIb / IIIa inhibitor. In some embodiments the from the drying step provides rehydrated platelets or throm GPIIb / IIIa inhibitor is GR144053 . In some embodiments, bosomes comprising from about 1 nM to about 1000 mM , GR144053 is present at a concentration of at least 1.2 uM . such as about 10 nM to about 10 mM , such as about 100 nM [0092 ] In some embodiments provided herein are drug to 1 mM , of the drug of step ( B ) . loaded platelets, drug - loaded platelet derivatives , or drug [ 0082 ] In some embodiments , the methods of preparing loaded thrombosomes prepared with an anti- aggregation drug - loaded platelets , drug - loaded platelet derivatives, or agent before being treated with the drug . In some embodi drug -loaded thrombosomes that comprise pooling platelets , ments provided herein are drug -loaded platelets , drug platelet derivatives , or thrombosomes from a plurality of loaded platelet derivatives , or drug - loaded thrombosomes donors comprise a viral inactivation step . prepared with an anti- aggregation agents concurrently with [0083 ] In some embodiments , the methods of preparing the drug. drug - loaded platelets , drug -loaded platelet derivatives, or [0093 ] In some embodiments provided herein are drug drug- loaded thrombosomes that comprise pooling platelets , loaded platelets , drug - loaded platelet derivatives, or drug platelet derivatives, or thrombosomes from a plurality of loaded thrombosomes prepared with a drug where the drug donors do not comprise a viral inactivation step . is a drug for the treatment of cancer . In some embodiments [ 0084 ] In some embodiments , the platelets , the platelet provided herein the cancer is hemangiosarcoma. derivatives, or the thrombosomes are loaded with the drug in [0094 ] In some embodiments provided herein the drug for a period of time of 5 minutes to 48 hours , such as 10 minutes the treatment of cancer is doxorubicin . In some embodi to 24 hours , such as 20 minutes to 12 hours , such as 30 ments , the drug for the treatment of hemangiosarcoma is minutes to 6 hours, such as 1 hour to 3 hours , such as about doxorubicin . 2 hours. In some embodiments , a concentration of drug from [0095 ] In some embodiments provided herein the cancer about 1 nM to about 1000 mM , such as about 10 nM to about includes hemangiosarcoma. In some embodiments provided 10 mM , such as about 100 nM to 1 mM , is loaded in a period herein the drug is a drug for the treatment of hemangiosar of time of 5 minutes to 48 hours , such as 10 minutes to 24 coma includes doxorubicin . hours, such as 20 minutes to 12 hours , such as 30 minutes [0096 ] In some embodiments provided herein the drug for to 6 hours , such as 1 hour minutes to 3 hours, such as about the treatment of cancer is . 2 hours . [0097 ] In some embodiments provided herein the drug for [0085 ] In some embodiments provided herein are drug the treatment of cancer is a PARP inhibitor. In some embodi loaded platelets , drug - loaded platelet derivatives , or drug ments provided herein the PAPR inhibitor is a loaded thrombosomes prepared by a method as disclosed [0098 ] Drug - loaded platelets , drug- loaded platelet deriva herein . In some embodiments provided herein are rehy tives , or drug - loaded thrombosomes may shield the drug drated platelets , platelet derivatives , or thrombosomes pre from exposure in circulation , thereby reducing or eliminat pared by a method as disclosed herein . ing systemic toxicity ( e.g. cardiotoxicity ) associated with the [0086 ] In some embodiments provided herein are drug drug . Drug - loaded platelets , drug - loaded platelet deriva loaded platelets , drug - loaded platelet derivatives , or drug tives, or drug - loaded thrombosomes may also protect the loaded thrombosomes prepared with Prostaglandin E1 drug from metabolic degradation or inactivation . Drug (PGE1 ) or Prostacyclin . In some embodiments provided delivery with drug- loaded platelets, drug- loaded platelet herein are drug - loaded platelets , drug - loaded platelet deriva derivatives , or drug - loaded thrombosomes may therefore be tives, or drug -loaded thrombosomes that are not prepared advantageous in treatment of diseases such as cancer, since with Prostaglandin E1 (PGE1 ) or Prostacyclin . drug- loaded platelets , drug- loaded platelet derivatives , or [0087 ] In some embodiments provided herein are drug drug -loaded thrombosomes facilitate targeting of cancer loaded platelets , drug - loaded platelet derivatives , or drug cells while mitigating systemic side effects . Drug - loaded loaded thrombosomes prepared with a chelating agent such platelets , drug - loaded platelet derivatives, or drug - loaded US 2020/0206143 A1 Jul . 2 , 2020 6

thrombosomes may be used in any therapeutic setting in [0113 ] FIG . 13B shows the effect of the addition of a which expedited healing process is required or advanta GPIIb /IIIa inhibitor , GR 144053, on the aggregation of geous. Potential applications include , for example , targeted platelets by 0 ( top ), 0.5 mg/ mL (middle ), or 1 mg /mL depletion of cancer cells with drugs. (bottom ) doxorubicin as measured by light transmittance . [0099 ] Accordingly, in some embodiments , provided [0114 ] FIG . 13C shows the effect of the addition of herein is a method of treating a disease as disclosed herein , Tirofiban on the aggregation of platelets by 0 ( top) or 0.65 comprising administering drug -loaded platelets , drug mg/ml (bottom ) doxorubicin . loaded platelet derivatives , or drug - loaded thrombosomes as [0115 ] FIG . 14 shows the effect of increasing concentra disclosed herein . Accordingly, in some embodiments, pro tion of a PARP inhibitor (olaparib ) on the total number of vided herein is a method of treating a disease as disclosed platelets loaded with PARPi and the amount of PARPI herein , comprising administering cold stored , room tem loaded . perature stored , cryopreserved thawed , rehydrated , and /or [ 0116 ] FIG . 15 shows the total drug load (PARP inhibitor ) lyophilized platelets , platelet derivatives, or thrombosomes increase following incubation of platelets with increasing as disclosed herein . In some embodiments , the disease is concentration of drug . cancer . [0117 ] FIG . 16 shows punctate intracellular staining of fluorescently tagged PARP inhibitor (80 uM ) after incuba DESCRIPTION OF DRAWINGS tion with platelets for 3 h . [0100 ] FIG . 1 shows resulting amounts of doxorubicin [0118 ] FIG . 17 shows the release of DOX from within load in platelets as a function of incubation time when platelets in response to strong platelet activating agents after evaluated by flow cytometry . cryopreservation for up to 7 days. Day 2 is plotted on the left [0101 ] FIG . 2 shows resulting amounts of doxorubicin and Day 7 is plotted on the right for each group . load in platelets following ADP and TRAP simulation when [0119 ] FIG . 18 shows DOX - loaded thrombosomes (mg evaluated by flow cytometry . Plt ) prepared by incubation with 600 uM Dox . Pre - lyo is [ 0102 ] FIG . 3 shows fluorescence intensity as measured during preparation before lyophilization and post - lyo is after by flow cytometry after platelet incubation with DOX lyophilization and rehydration of the DOX - loaded Throm encapsulated liposomes. bosomes . [0103 ] FIGS. 4A -4C shows flow cytometry data relating [0120 ] FIG . 19 shows that the intracellular DOX concen to the endocytic loading of DOX into platelets . The top tration is increased 50 - fold prior to lyophilization (platelets ) graph shows amalgamated data that includes the bottom left and maintained at 30 - fold after lyophilization (thrombo graph (Doxorubicin at 0.1 mM ) and the bottom right graph somes ) relative to the external DOX concentration during (Doxorubicin at 0.3 mm ). incubation . [0104 ] FIG . 5 provides flow cytometry data relating to [0121 ] FIG . 20 shows platelet counts according to AcT liposome- mediated loading of Doxorubicin into platelets . Diff hematology analyzer remain unchanged after [0105 ] FIGS. 6A and 6B provide a comparison of doxo some (liposome encapsulated doxorubicin ) loading effi lyophilization ( thrombosomes ). Unloaded Thrombosomes is ciency in platelets between conventional HMT buffer (Pro plotted on the left and DOX - loaded Thrombosomes is plot tocol 3 , shown in continuous line ) and trehalose -containing ted to the right for each group . loading buffer (Protocol 4 , shown in individual points ). FIG . [0122 ] FIG . 21 shows that DOX loaded thrombosomes 6A shows platelets with CD42b antibodies, while FIG . 6B have similar platelet receptor biomarker expression relative shows platelets without CD42b to unloaded thrombosomes immediately prior to (platelets ) [0106 ] FIG . 7 shows the correlation between fluorescence and after lyophilization ( thrombosomes ). and concentration of doxorubicin . [0123 ] FIG . 22 shows DOX - loaded thrombosomes adhere [0107 ] FIG . 8 shows the total concentration of ( a ) intrac to collagen and tissue factor coated microchips similar to ellular doxorubicin ( triangle ) , and ( b ) membrane -bound unloaded thrombosomes . doxorubicin (circle ), with increasing concentration of plate [ 0124 ] FIG . 23 shows DOX - loaded thrombosomes gener lets /?L . ate thrombin similar to unloaded thrombosomes . [0108 ] FIG . 9 shows how for a given concentration of [0125 ] FIG . 24 shows the effect of DOX - loaded throm doxorubicin (DOX ) (0.2 mm ), the amount of DOX /platelet bosomes on cell growth in a hemangiosarcoma cell model decreases as the number of platelets increases . using different amounts of DOX - loaded and unloaded [0109 ] FIG . 10 shows the effect of collagen on inducing thrombosomes . drug release from doxorubicin -loaded platelets . Released [0126 ] FIG . 25A shows that paclitaxel loads into platelets DOX is plotted on the left and intracellular DOX is plotted in a dose -dependent manner as measured by flow cytometry . to the right for each group . 200 uM / 4 uM is plotted on the bottom and 400 uM / 8 uM is [0110 ] FIG . 11 shows the effect of TRAP - 6 , ADP , arachi plotted on top donic acid , and thrombin on inducing drug release from [0127 ] FIG . 25B shows that the percent of platelets loaded doxorubicin - loaded platelets . Released DOX is plotted on with paclitaxel increases with increasing concentration of the left and intracellular DOX is plotted to the right for each paclitaxel over time. 200 uM / 4 uM is plotted on the bottom group . and 400 uM / 8 uM is plotted on top . [ 0111 ] FIG . 12 shows a comparison of the amount of [0128 ] FIGS . 26A - C shows that platelet counts remain doxorubicin loaded into platelets with loading buffer ( right) more stable as a function of a higher initial starting cell vs. the amount loaded with HMT buffer (left ) . count and minimally 50 % are retained after loading . PTX in [0112 ] FIG . 13A shows the effect of DOX on the aggre 1 % DMSO is plotted on the bottom , PTX in 5 % DMSO is gation of platelets as measured by light transmittance . plotted in the middle and unloaded is plotted on top . US 2020/0206143 A1 Jul . 2 , 2020 7

[0129 ] FIG . 27 shows brightfield , fluorescence , and over than about 5.0 um ( e.g., less than about 2.5 um , less than laid microscope images of platelets loaded with fluores about 2.0 um , less than about 1.5 um , less than about 1.0 um , cently labeled Paclitaxel. less than about 0.9 um , less than about 0.8 um , less than about 0.7 um , less than about 0.6 um , less than about 0.5 um , DETAILED DESCRIPTION less than about 0.4 um , or less than about 0.3 um ). In some [0130 ] Unless defined otherwise, all technical and scien embodiments , the particle size is from about 0.3 um to about tific terms used herein have the same meaning as commonly 5.0 um ( e.g. , from about 0.4 um to about 4.0 um , from about understood by one of ordinary skill in the art to which the 0.5 um to about 2.5 um , from about 0.6 um to about 2.0 um , term belongs. Although any methods and materials similar from about 0.7 um to about 1.0 um , from about 0.5 um to or equivalent to those described herein can be used in the about 0.9 um , or from about 0.6 um to about 0.8 um ) . practice or testing of the present disclosure , the preferred [0137 ] In some embodiments , at least 50 % ( e.g., at least methods and materials are now described . All publications about 55 % , at least about 60 % , at least about 65 % , at least mentioned herein are incorporated herein by reference to about 70 % , at least about 75 % , at least about 80 % , at least disclose and describe the methods and/ or materials in con about 85 % , at least about 90 % , at least about 95 % , or at least nection with which the publications are cited . The present about 99 % ) of platelets and /or the dried platelets , such as disclosure is controlling to the extent it conflicts with any freeze -dried platelets , have a particle size in the range of incorporated publication . about 0.3 um to about 5.0 um (e.g. , from about 0.4 um to [0131 ] As used herein and in the appended claims, the about 4.0 um , from about 0.5 um to about 2.5 um , from term “ platelet ” can include whole platelets , fragmented about 0.6 um to about 2.0 um , from about 0.7 um to about platelets , platelet derivatives, or thrombosomes . Thus, for 1.0 um , from about 0.5 um to about 0.9 um , or from about example , reference to " drug - loaded platelets ” may be inclu 0.6 um to about 0.8 um ). In some embodiments , at most 99 % sive of drug - loaded platelets as well as drug - loaded platelet (e.g. , at most about 95 % , at most about 80 % , at most about derivatives or drug - loaded thrombosomes, unless the con 75 % , at most about 70 % , at most about 65 % , at most about text clearly dictates a particular form . 60 % , at most about 55 % , or at most about 50 % ) of platelets [0132 ] As used herein and in the appended claims, the and /or the dried platelets , such as freeze -dried platelets , are singular forms “ a ” , “ an ” , and “ the” include plural referents in the range of about 0.3 um to about 5.0 um ( e.g., from unless the context clearly dictates otherwise. Thus , for about 0.4 um to about 4.0 um , from about 0.5 um to about example , reference to " a platelet ” includes a plurality of 2.5 um , from about 0.6 um to about 2.0 um , from about 0.7 such platelets . Furthermore , the use of terms that can be um to about 1.0 um , from about 0.5 um to about 0.9 um , or described using equivalent terms include the use of those from about 0.6 um to about 0.8 um ) . In some embodiments , equivalent terms. Thus, for example , the use of the term about 50 % to about 99 % ( e.g. , about 55 % to about 95 % , “ subject ” is to be understood to include the terms “ patient” , about 60 % to about 90 % , about 65 % to about 85 , about 70 % “ individual” and other terms used in the art to indicate one to about 80 % ) of platelets and /or the dried platelets , such as who is subject to a treatment. freeze -dried platelets , are in the range of about 0.3 um to [0133 ] In some embodiments , rehydrating the drug - loaded about 5.0 um ( e.g., from about 0.4 um to about 4.0 um , from platelets comprises adding to the platelets an aqueous liquid . about 0.5 um to about 2.5 um , from about 0.6 um to about In some embodiments , the aqueous liquid is water. In some 2.0 um , from about 0.7 um to about 1.0 um , from about 0.5 embodiments , the aqueous liquid is an aqueous solution . In um to about 0.9 um , or from about 0.6 um to about 0.8 um ). some embodiments , the aqueous liquid is a saline solution . [0138 ] In some embodiments , platelets are isolated prior In some embodiments , the aqueous liquid is a suspension . to treating the platelets with a drug . [0134 ] In some embodiments , the rehydrated platelets [0139 ] Accordingly , in some embodiments , the method for have coagulation factor levels showing all individual factors preparing drug- loaded platelets comprises : ( e.g., Factors VII , VIII and IX ) associated with blood [0140 ] a ) isolating platelets , for example in a liquid clotting at 40 international units ( IU ) or greater. medium ; [0135 ] In some embodiments , the dried platelets , such as [0141 ] and freeze -dried platelets , have less than about 10 % , such as less [0142 ] b ) treating the platelets with a drug and with a than about 8 % , such as less than about 6 % , such as less than loading buffer comprising a salt, a base , a loading about 4 % , such as less than about 2 % , such as less than about agent, and optionally ethanol, to form the drug -loaded 0.5 % crosslinking of platelet membranes via proteins and /or platelets . lipids present on the membranes . In some embodiments , the [0143 ] Accordingly , in some embodiments , the method for rehydrated platelets , have less than about 10 % , such as less preparing drug- loaded platelets comprises : than about 8 % , such as less than about 6 % , such as less than [0144 ] a ) isolating platelets , for example in a liquid about 4 % , such as less than about 2 % , such as less than about medium ; 0.5 % crosslinking of platelet membranes via proteins and /or [0145 ] b ) treating the platelets with a drug to form a first lipids present on the membranes . composition , and [ 0136 ] In some embodiments , the drug -loaded platelets ( 0146 ] c ) treating the first composition with a buffer and the dried platelets, such as freeze- dried platelets, having comprising a salt, a base, a loading agent, and option a particle size ( e.g. , diameter , max dimension ) of at least ally at least one organic solvent to form the drug - loaded about 0.2 um ( e.g., at least about 0.3 um , at least about 0.4 platelets. um , at least about 0.5 um , at least about 0.6 um , at least [0147 ] In some embodiments , suitable organic solvents about 0.7 um , at least about 0.8 um , at least about 0.9 um , include , but are not limited to alcohols , esters , ketones, at least about 1.0 um , at least about 1.0 um , at least about 1.5 ethers, halogenated solvents , hydrocarbons , nitriles, glycols , um , at least about 2.0 um , at least about 2.5 um , or at least alkyl nitrates, water or mixtures thereof. In some embodi about 5.0 um ). In some embodiments , the particle size is less ments , suitable organic solvents includes , but are not limited US 2020/0206143 A1 Jul . 2 , 2020 8

to methanol, ethanol, n - propanol, isopropanol , acetic acid , [0169 ] In some embodiments , platelets are derived in acetone , methyl ethyl ketone , methyl isobutyl ketone , vitro . In some embodiments , platelets are derived or pre methyl acetate , ethyl acetate , isopropyl acetate , tetrahydro pared in a culture prior to treating the platelets with a drug . furan , isopropyl ether (IPE ) , tert -butyl methyl ether , dioxane In some embodiments , preparing the platelets comprises ( e.g. , 1,4 -dioxane ) , acetonitrile , propionitrile , methylene deriving or growing the platelets from a culture of mega chloride, chloroform , toluene, anisole , cyclohexane, hexane, karyocytes . In some embodiments, preparing the platelets heptane , ethylene glycol, nitromethane , dimethylforma comprises deriving or growing the platelets ( or megakaryo mide, dimethyl sulfoxide , N -methyl pyrrolidone , dimethyl cytes ) from a culture of human pluripotent stem cells acetamide , and combinations thereof. (PCSs ) , including embryonic stem cells (ESCs ) and /or [0148 ] Accordingly , in some embodiments , the method for induced pluripotent stem cells (iPSCs ) . preparing drug - loaded platelets comprises : [0170 ] Accordingly , in some embodiments , themethod for [0149 ] a ) isolating platelets , for example in a liquid preparing drug -loaded platelets comprises : medium ; [0150 ] b ) treating the platelets with a buffer comprising [0171 ] a ) preparing platelets ; a salt , a base , a loading agent, and optionally at least [0172 ] and one organic solvent, to form a first composition ; and [0173 ] b ) treating the platelets with a drug and with a [0151 ] c ) treating the first composition with a drug , to loading buffer comprising a salt, a base , a loading form the drug - loaded platelets . agent , and optionally at least one organic solvent, to [0152 ] In some embodiments , no solvent is used . Thus , in form the drug - loaded platelets . some embodiments , the method for preparing drug - loaded (0174 ] Accordingly , in some embodiments , the method for platelets comprises: preparing drug- loaded platelets comprises : [0153 ] a ) isolating platelets , for example in a liquid [0175 ] a ) preparing platelets ; medium ; [0176 ] b ) treating the platelets with a drug to form a first [0154 ] and composition ; and [0155 ] b ) treating the platelets with a drug and with a loading buffer comprising a salt , a base , and a loading [0177 ] c ) treating the first composition with a buffer agent , to form the drug - loaded platelets , comprising a salt , a base, a loading agent, and option [0156 ] wherein the method does not comprise treat ally at least one organic solvent, to form the drug ing the platelets with an organic solvent such as loaded platelets . ethanol. [ 0178 ] Accordingly , in some embodiments , the method for [0157 ] Thus, in some embodiments , the method for pre preparing drug- loaded platelets comprises : paring drug - loaded platelets comprises : [0179 ] a ) preparing platelets ; [ 0158 ] a ) isolating platelets , for example in a liquid [0180 ] b ) treating the platelets with a buffer comprising medium ; a salt, a base , a loading agent, and optionally at least [0159 ] b ) treating the platelets with a drug to form a first one organic solvent, to form a first composition ; and composition ; and [ 0181 ] c ) treating the first composition with a drug , to [0160 ] c ) treating the first composition with a buffer form the drug - loaded platelets . comprising a salt, a base , and a loading agent, to form [0182 ] In some embodiments , no solvent is used . Thus, in the drug - loaded platelets , some embodiments , the method for preparing drug -loaded [0161 ] wherein the method does not comprise treat platelets comprises: ing the platelets with an organic solvent such as ethanol and the method does not comprise treating [0183 ] a ) preparing platelets ; the first composition with an organic solvent such as [ 0184 ) and ethanol. [ 0185 ] b ) treating the platelets with a drug and with a [0162 ] Thus, in some embodiments , the method for pre loading buffer comprising a salt , a base , and a loading paring drug- loaded platelets comprises : agent, to form the drug - loaded platelets , [0163 ] a ) isolating platelets , for example in a liquid (0186 ] wherein the method does not comprise treat medium ; ing the platelets with an organic solvent such as [0164 ] b ) treating the platelets with a buffer comprising ethanol. a salt, a base , and a loading agent , to form a first [0187 ] Thus , in some embodiments , the method for pre composition ; and paring drug - loaded platelets comprises: [0165 ] c ) treating the first composition with a drug , to form the drug - loaded platelets . [0188 ] a ) preparing platelets ; [0166 ] wherein the method does not comprise treat [0189 ] b ) treating the platelets with a drug to form a first ing the platelets with an organic solvent such as composition ; and ethanol and the method does not comprise treating [0190 ] c ) treating the first composition with a buffer the first composition with an organic solvent such as comprising a salt, a base , and a loading agent , to form ethanol. the drug - loaded platelets , [0167 ] In some embodiments , isolating platelets com [0191 ] wherein the method does not comprise treat prises isolating platelets from blood . ing the platelets with an organic solvent such as [0168 ] In some embodiments , platelets are donor- derived ethanol and the method does not comprise treating platelets . In some embodiments , platelets are obtained by a the first composition with an organic solvent such as process that comprises an apheresis step . ethanol. US 2020/0206143 A1 Jul . 2 , 2020 9

[0192 ] Thus, in some embodiments , the method for pre [0203 ] miRNAs and siRNAs are distinct from other types paring drug -loaded platelets comprises: of RNA molecules including, without limitation ,messenger [0193 ] a ) preparing platelets ; RNA (“ mRNA ), ribosomal RNA (“ rRNA " ) , small nuclear [0194 ] b ) treating the platelets with a buffer comprising RNA (“ snRNA ” ) , transfer RNA ( “ RNA ” ) , and short hairpin a salt, a base , and a loading agent, to form a first RNA ( “ shRNA " ) . rRNA , SRNA , TRNA , and shRNA are all composition ; and encompassed within the term “ drug ” as used herein .mRNA , [0195 ] c ) treating the first composition with a drug, to rRNA , SRNA , and tRNA are canonical classes of RNA form the drug - loaded platelets . molecules, the function and structure of which are well [0196 ] wherein the method does not comprise treat known to those of ordinary skill in the art . ing the platelets with an organic solvent such as [0204 ] shRNAs are short linear RNA molecules , portions ethanol and the method does not comprise treating of which associate with each other via base pairing to form the first composition with an organic solvent such as a double stranded stem region ( as opposed to the fully ethanol . double stranded siRNAs ), resulting in a characteristic “ hair [0197 ] In some embodiments , the loading agent is a sac pin ” or loop at one end of the molecule . Unlike miRNAs and charide. In some embodiments , the saccharide is a mono siRNAs , shRNAs are typically introduced into cells using saccharide . In some embodiments, the saccharide is a disac methods that differ from the methods used for introducing charide. In some embodiments , the saccharide is a non miRNA and siRNA ( e.g., using transfection methods) . For reducing disaccharide. In some embodiments , the saccharide example , shRNAs can be introduced via plasmids or, alter is sucrose ,maltose , trehalose , glucose ( e.g., dextrose ) ,man natively , through viral or bacterial vectors. Both of these nose , or xylose . In some embodiments , the loading agent is methods are DNA -based techniques, where the shRNA is a starch . transcribed , processed by the enzyme Drosha , and then [ 0198 ] In some embodiments , the loading agent is a carrier further processed into siRNAs, by the Dicer enzyme, to protein . In some embodiments , the carrier protein is albu eventually mediate RNAi. min . In some embodiments , the carrier protein is bovine [0205 ] As used herein , “ thrombosomes” ( sometimes also serum albumin ( BSA ) . herein called “ Tsomes” or “ Ts” , particularly in the Examples [0199 ] As used herein , the term “ drug ” refers to any agent and Figures) are platelet derivatives that have been treated suitable for the treatment of cancer other than a messenger with an incubating agent ( e.g. , any of the incubating agents RNA (mRNA ) , a microRNA ( also known as miRNA ) and /or described herein ) and lyopreserved ( e.g., freeze- dried ). In a small interfering RNA ( also known as siRNA, short some cases, thrombosomes can be prepared from pooled interfering RNA , or silencing RNA ) . platelets. Thrombosomes can have a shelf life of 2-3 years [0200 ] As used herein , the term “ mRNA ” refers to a in dry form at ambient temperature and can be rehydrated single - stranded ribonucleic acid molecule used by cells for with sterile water within minutes for immediate infusion . the translation of DNA into protein . Many mRNAs are [0206 ] In some embodiments , the drug is selected from the naturally occurring , but mRNAs can also be synthesized by group consisting of one of the following : those of ordinary skill in the art. Mature mRNAs can vary [0207 ] i . a smallmolecule ( that is , an organic compound greatly in size and composition . mRNAs are necessary having a molecular weight up to about 2 KDalton ); components of protein synthesis after exportation from the [ 0208 ] ii. a protein ; nucleus to the cytoplasm of the cell . [0209 ] iii . an oligopeptide; [ 0201 ] As used herein , the term “ microRNA ” refers to a [ 0210 ] iv . a non -miRNA nucleic acid , a non -siRNA , ribonucleic acid duplex that targets and silences an mRNA and /or a non -mRNA ( e.g., non -miRNA , DNA , other molecule. Many miRNAs are naturally -occurring , but miR naturally or non - naturally occurring nucleic acids, NAs can also be synthesized by those of ordinary skill in the including various modifications thereof) ; art. Mature miRNAs are generally 19-25 nucleotides in [0211 ] and length , have 3 ' overhangs of two nucleotides, target multiple [ 0212 ] V. an aptamer . mRNAs and are typically only partially complementary to [0213 ] In various methods described herein , platelets are their target mRNAs. miRNAs typically function by repress loaded with one or more any of a variety of drugs . In some ing translation and facilitating mRNA degradation . embodiments , platelets are loaded with a small molecule . [0202 ] As used herein , the term “ small interfering RNA ” For example , platelets can be loaded with one or more of refers to a double -stranded RNA that targets and silences an vemurafenib (ZELBORAF® ), dabrafenib ( TAFINLAR® ), mRNA molecule . Many siRNAs are naturally -occurring , but encorafenib (BRAFTOVITM ) , BMS - 908662 (XL281 ) , siRNAs can also be synthesized by those of ordinary skill in sorafenib , LGX818 , PLX3603 , RAF265 , RO5185426 , the art . siRNA are generally derived from strands of exog GSK2118436 , ARQ 736 , GDC -0879 , PLX - 4720 , AZ304 , enous growing (originating from outside an organism ) RNA , PLX - 8394 , HM95573 , RO5126766 , LXH254 , trametinib which is taken up by the cell and undergoes further pro (MEKINIST® , GSK1120212) , cobimetinib ( COTEL cessing. Mature siRNAs are generally 19-27 nucleotides in LIC® ), binimetinib (MEKTOVI® , MEK162 ) , selumetinib length , have 3 ' overhangs of two nucleotides at each end that ( AZD6244 ) , PD0325901 , MSC1936369B , SHR7390 , TAK can be used to “ interfere ” with the translation of proteins by 733 , CS3006 , WX - 554 , PD98059 , C11040 (PD184352 ) , binding to and promoting the degradation of messenger hypothemycin , FRI- 20 (ON - 01060 ), VTX - Ile , 25 -OH - D3 RNA at specific sequences . Each siRNA strand has a 5 ' 3 -BE ( B3CD , bromoacetoxycalcidiol) , FR - 180204 , AEZ phosphate group and a 3 ' hydroxyl (OH ) group . siRNA can 131 (AEZS - 131) , AEZS -136 , AZ - 13767370 , BL - EI- 001 , be produced from dsRNA or hairpin looped RNA and LY - 3214996 , LTT- 462 , KO - 947 , KO - 947 , MK -8353 processed into siRNAsby the Dicer enzyme. siRNA can also ( SCH900353 ) , SCH772984 , ulixertinib (BVD -523 ) , be incorporated into a multi -subunit protein complex called CC - 90003 , GDC -0994 (RG -7482 ) , ASNO07 , FR148083 , RNA - induced silencing complex (RISC ) . 5-7 -oxozeaenol , 5 - iodotubercidin , GDC0994 , ONC201, US 2020/0206143 A1 Jul . 2 , 2020 10 buparlisib (BKM120 ) , (BYL719 ) , WX -037 , copan , , olaparib , , iniparib , ruca lisib (ALIQOPATM , BAY80-6946 ) , dactolisib (NVP parib , CEP - 9722 , E7016 , E7449, PRN1371, BLU9931 , BEZ235 , BEZ - 235 ) , taselisib (GDC -0032 , RG7604 ), sono FIIN - 4 , H3B -6527 , NVP - BGJ398 , ARQ087, TAS - 120 , lisib (PX -866 ), CUDC -907 , PQR309 , ZSTK474 , SF1126 , CH5183284 , Debio 1347 , INCB054828 , JNJ- 42756493 (er AZD8835, GDC -0077 , ASNO03, pictilisib (GDC -0941 ) , dafitinib ), rogaratinib (BAY1163877 ) , FIIN - 2 , LY2874455 , pilaralisib ( XL147 , SAR245408 ) , gedatolisib (PF lenvatinib (E7080 ), ponatinib (AP24534 ) , regorafenib (BAY 05212384 , PKI- 587 ), serabelisib ( TAK -117 , MLN1117 , 73-4506 ), dovitinib ( TK1258 ), lucitanib (E3810 ), cediranib INK 1117) , BGT- 226 (NVP -BGT226 ) , PF - 04691502 , api ( AZD2171) , nintedanib (OFEV® , BIM 1120 ) , brivanib tolisib (GDC -0980 ) , omipalisib (GSK2126458 , GSK458 ) , (BMS - 540215 ), ASP5878 , AZD4547 , BGJ398 ( infigra voxtalisib (XL756 , SAR245409 ), AMG 511, CH5132799 , tinib ), E7090 , HMPL -453 , MAX -40279 , XL999, orantinib GSK1059615 , GDC -0084 (RG7666 ) , VS -5584 (SB2343 ) , (SU6668 ), pazopanib (VOTRIENT® ), anlotinib , AL3818, PKI- 402 , wortmannin , LY294002 , PI- 103 , rigosertib , PRIMA - 1 (p53 reactivation induction of massive apoptosis XL - 765 , LY2023414 , SAR260301, KIN - 193 (AZD -6428 ), 1 ), APR - 246 (PRIMA -IMET ) , 2 -sulfonylpyrimidines such GS -9820 , AMG319, GSK2636771 , NL -71-101 , H - 89 , as PK11007, pyrazoles such as PK7088 , zinc metallochap GSK690693 , CCT128930 , AZD5363 , ipatasertib (GDC erone - 1 (ZMC1 ; NSC319726 / ZMC 1 ) , a thiosemicarbazone 0068 , RG7440 ), A -674563 , A -443654 , AT7867 , AT13148 , ( e.g., COTI- 2 ), CP -31398 , STIMA - 1 (SH Group - Targeting uprosertib , afuresertib , DC120 , 2- [4- ( 2 - aminoprop - 2 - yl ) Compound That Induces Massive Apoptosis ), MIRA - 1 phenyl] -3 -phenylquinoxaline , MK - 2206 , edelfosine, erucyl (NSC19630 ) and its analogs, e.g., MIRA - 2 and MIRA -3 , phophocholine , erufosine , SR13668, OSU - A9, PH - 316 , RITA (NSC652287 ) , chetomin (CTM ) , stictic acid PHT- 427 , PIT - 1 , DM -PIT - 1, triciribine ( triciribine phos (NSC87511 ) , p53R3, SCH529074 , WR - 1065, arsenic com phate monohydrate ), API- 1, N-( 4- ( 5- ( 3 - acetamidophenyl) pounds, gambogic acid , spautin - 1, YK - 3-237 , NSC59984 , 2-( 2 - aminopyridin - 3 - yl) -3H - imidazo [ 4,5 - b ] pyridin - 3 - yl) disulfiram (DSF ), G418 , RETRA ( reactivate transcriptional benzyl) -3 - fluorobenzamide , ARQ092 , BAY 1125976 , activity ), PD0166285 , 17- AAG , geldanamycin , ganetespib , 3 -oxo -tirucallic acid , lactoquinomycin , boc- Phe -vinyl AUY922, IPI- 504 , / SAHA , /depsipep ketone, Perifosine ( D -21266 ) , TCN , TCN - P, GSK2141795, tide, HB1-8000, RG7112 (RO5045337 ) , RO5503781 , MLN0128 , AZD -2014 , CC -223 , AZD2014 , CC - 115 , MI- 773 (SAR405838 ), DS -3032b , AM - 8553 , AMG 232 , (RAD001 ) , temsirolimus ( CCI- 779 ) , ridaforoli MI- 219, MI- 713 , MI- 888 , TDP521252 , NSC279287 , mus (AP - 23573 ), , BMS- 214662, L778123 , PXN822 , ATSP - 7041, spiroligomer , PK083 , PK5174 , L744832 , FTI- 277 , PRI- 724 , CWP232291 , PNU74654 , PK5196 , nutlin 3a , RG7388 , Ro- 2443 , FTY - 720 , ceramide , PKF115-584 , PKF118-744 , PKF118-310 PFK222-81 OP449 , vatalanib (PTK787 / ZK222584 ) , TKI- 538 , sunitinib CGP 049090 , ZTM000990 , BC21, methyl 3 - {[ (4 -methyl (SU11248 , SUTENT® ), thalidomide, lenalidomide (REV phenyl) sulfonyl ) amino } benzoate (MSAB ), AV65 , iCRT3 , LIMID® ) , axitinib ( AG013736 , INLYTA® ) , RXC0004 , iCRT5 , iCRT14 , SM04554 , LGK 974, XAV939 , curcumin ETC - 159, LGK974 , WNT- 059 , AZD8931 , AST1306 , ( e.g., Meriva® ) , DIF - 1 , genistein , NSC668036 , FJ9 , BML CP724714 , CUDC101, TAK285 , AC480 , DXL -702 , E -75 , 286 ( 3289-8625 ) , IWP, IWP - 1 , IWP - 2 , JW55 , G007- LK , PX - 104.1 , ZW25 , CP- 724714 , irbinitinib ( ARRY - 380 , pyrvinium , foxy - 5 , Wnt- 5a , ipafricept (OMP - 54F28 ), van ONT- 380 ) , TAS0728 , AST- 1306 , AEE -788 , perlitinib tictumab (OMP - 18R5 ) , SM04690 , SM04755 , nutlin - 3a , (EKB -569 ) , PKI- 166 , D -69491 , HKI- 357 , AC -480 (BMS IWR1, JW74 , okadaic acid , SB239063 , SB203580 , adenos 599626 ) . RB - 200h , ARRY - 334543 ( ARRY - 543 , ine diphosphate (hydroxymethyl ) pyrrolidinediol (ADP ASLAN001 ), CUDC - 101 , IDM -1 , , cytosine ara HPD ), 2- [4- ( 4 - fluorophenylpiperazin - 1 -yl ) -6 -methylpy binoside , ORY1001 (RG6016 ) , GSK2879552 , rimidin -4 (3H ) -one , PJ34 , J01-017a , IC261, PF670462, INCB059872 , IMG7289 , CC90011, MI1, M12 , MI3 , Mi2-2 bosutinib (BOSULIFR ) , PHA665752 , imatinib (MI - 2-2 ) , M1463 ,M1503 , MIV -6R , EPZ004777, EPZ -5676 , (GLEEVEC® ) , ICG - 001, Rp - 8 - Br- CAMP , SDX - 308 , SGC0946 , CN - SAH , SYC -522 , SAH , SYC -534 , MM - 101 , WNT974 , CGX1321 , ETC - 1922159 , AD -REIC / Dkk3, MM - 102 , MM - 103 , MM - 401 , WDR5-0101, WDR5-0102 , WIKI4 , windorphen , NTRC 0066-0 , CFI- 402257 , a (5,6 WDR5-0103 , OICR -9429 , tivantinib ( ARQ 197) , golvatinib dihydro )pyrimido [4,5 -ejindolizine , BOS172722 , 563845 , ( E7050 ) , cabozantinib (XL 184 , BMS - 907351 ) , foretinib AZD5991 , AMG 176 , 483 - LM , MIK665 , TASIN - 1 ( Trun (GSK1363089 ), crizotinib (PF -02341066 ), MK - 2461, BPI cated APC Selective Inhibitor) , osimertinib ( AZD9291, 9016M , TQ - B3139 , MGCD265, MK -8033 , capmatinib merelectinib , TAGRISSOTM ), erlotinib ( TARCEVA® ), gefi (INC280 , INCB28060 ), tepotinib (MSC2156119 ), tinib ( IRESSA® ) , neratinib (HKI - 272 , NERLYNX® ), lapa EMD1214063 ) , CE - 35562 , AMG - 337 , AMG -458 , PHA tinib ( TYKERB® ) , vetanib (CAPRELSA® ), rociletinib 665725 , PF -04217903 , SU11274 , PHA -665752 , HS - 10241 , (CO - 1686 ) , olmutinib (OLITATM , HM61713 , BI- 1482694 ) , ARGX -111 , glumetinib (SCC244 ), EMD 1204831, naquotinib (ASP8273 ), nazartinib (EGF816 , NVS -816 ) , AZD6094 ( savolitinib , volitinib , HMPL - 504 ), PLB1001, PF - 06747775 , icotinib ( BPI- 2009H ) , afatinib ( BIBW 2992 , ABT- 700 , AMG 208 , INCB028060, AL2846 , HTI- 1066 , GILOTRIF® ) , dacomitinib (PF -00299804 , PF - 804 , PF -299 , PT2385 , PT2977 , 17 allylamino -17 -demethoxygeldanamy PF - 299804 ) , avitinib ( AC0010 ), AC001OMA EA1045 , can cin , (HALAVEN® , E389 , ER -086526 ), ibrutinib ertinib (CI - 1033 ) , poziotinib (NOV120101 , HM781-36B ) , (PCI - 32675 , Imbruvica® ) ( 1 - [ ( 3R ) -3- [ 4 - amino - 3-( 4 - phe AV -412 , W24002 , brigatinib (AP26113 , ALUNBRIG® ), noxyphenyl )pyrazolo [3,4 - d ]pyrimidin - 1- yl] piperidin - 1- yl] pelitinib (EKB - 569 ) , tarloxotinib ( TH -4000 , PR610 ) , BPI prop - 2 - en - 1 - one) ; AC0058 (AC0058TA ) ; N-( 3 - ( (2- ( 13 15086 , Hemay022 , ZN - e4 , tesevatinib (KDO19 , XL647 ) , fluoro -4- (4 -methylpiperazin -1 -yl ) phenyl) amino ) -7H YH25448 , epitinib (HMPL - 813 ) , CK - 101, MM - 151, pyrrolo [ 2,3- d ]pyrimidin - 4 - yl) oxy ) phenyl) acrylamide ; AZD3759 , ZD6474 , PF -06459988 , varlintinib (ASLAN001 , acalabrutinib (ACP - 196 , Calquence , INN ) ( 4-[ 8 -amino ARRY -334543 ), AP32788 , HLX07 , D -0316 , AEE788 , 3 -[ (2S ) -1- but - 2- ynoylpyrrolidin -2 - yllimidazo [ 1,5 -a ] HS- 10296 , GW572016 , pyrotinib ( SHR1258 ), , pyrazin - 1 -yl ) -N -pyridin - 2 -ylbenzamide ) ; zanubrutinib US 2020/0206143 A1 Jul . 2 , 2020 11

( BGB - 3111 ) (( 7R )-2- ( 4 - phenoxyphenyl) -7- ( 1- prop -2 ( OGIVRI® ) , ado - trastuzumab emtansine (KADCYLA? , enoylpiperidin - 4 -yl ) -1,5,6,7 - tetrahydropyrazolo [ 1,5 - a ]py T -DM1 ) , Zemab , DS- 8201a , MFGR1877S , B -701 , rilotu rimidine - 3 - carboxamide ) ; spebrutinib ( AVL -292 , 1202757 mumab (AMG102 ), ficlatuzumab (AV - 299 ), FP - 1039 89-8 , Cc - 292) (N- [ 3 -[ [5 - fluoro - 2-[ 4- ( 2 -methoxyethoxy ) (GSK230 ) , TAK701, YYB101, onartuzumab (MetMAL ) , anilino ]pyrimidin -4 - yl ] amino ]phenyl ]prop - 2- enamide) ; ipilimumab (YERVOY® ) , tremelimumab (CP -675,206 ), poseltinib (HM71224 , LY3337641) (N- [ 3- [ 2-[ 4- ( 4 -methyl pembrolizumab (KEYTRUDA® ), nivolumab (OPDIVO? ), piperazin - 1 - yl) anilino ] furo [ 3,2- d ]pyrimidin - 4 - yl ]oxyphe atezolizumab ( TECENTRIQ® ) , avelumab (BAVENCIO? ) , nyl] prop - 2 -enamide ) ; evobrutinib (MSC 2364447 , M -2951 ) durvalumab ( IMFINZITM ), BIOO - 1 , BIOO - 2 , and BIOO - 3 . ( 1- [ 4 - [ [ [6 -amino - 5-( 4 -phenoxyphenyl ) pyrimidin - 4 - yl] [ 0215 ] In various methods described herein , platelets are amino ]methyl ] piperidin - 1 -yl ] prop - 2 - en - 1 - one ) ; tirabrutinib loaded with one or more any of a variety of drugs. In some ( ONO -4059 , GS - 4059 , ONO /GS -4059 , ONO -WG - 307 ) ( 1 embodiments , platelets are loaded with an oligopeptide . For [ 4 -[ [[ 6 -amino -5- ( 4 -phenoxyphenyl )pyrimidin - 4 -yl ] amino ] example , platelets can be loaded with one or more of methyl ]piperidin - 1 - yl ] prop - 2 -en - 1 - one ); vecabrutinib RGD -SSL - Dox , LPD -PEG -NGR , PNC - 2 , PNC - 7 , RGD ( SNS- 062 ) ( ( 3R ,4S ) -1-( 6 - amino - 5 - fluoropyrimidin - 4 - yl) -3 PEG - Suc - PD0325901, VWCS , FWCS, p16 , Bac - 7 - ELP [( 3R )-3- (3 -chloro -5- (trifluoromethyl ) anilino ) -2 -oxopiperi p21, Pen - ELP -p21 , TAT- Bim , Poropeptide - Bax , R8 - Bax , din - 1- yl ]piperidine - 4 - carboxamide ); dasatinib (Sprycel® ; CT20p -NP , RRM -MV , RRM - IL12 , PNC - 27 , PNC -21 , PNC BMS- 354825 ) ( N - 2 - chloro - 6 -methylphenyl ) -2 - [ [6- [4- ( 2 28 , Tat - aHDM2, Int- H1 - S6A , FBA , Pen -ELP -H1 , BAC1 hydroxyethyl) piperazin - 1 - yl] -2 -methylpyrimidin - 4 - yl] ELP -H1 , goserelin , leuprolide , Buserelin , Triptorelin , amino ]-1,3 - thiazole- 5 -carboxamide ) ; PRN1008 , PRN473 , Degarelix , Pituitary adenylate cyclase activating ABBV - 105 , CG'806 , ARQ 531, BIIB068 , AS871, CB1763 , (PACAP ), cilengitide , ATN - 161 ( AcPHSCN -NH2 ) , TTK , CB988 , GDC - 0853 , RN486 , GNE -504 , GNE- 309, BTK LYOK , IMP - 3 , P16_37-63 , VEGFR1- A24-1084 , uMMP- 2 , Max , CT- 1530 , CGI- 1746 , CGI- 560 , LFM A13 , TP -0158 , UTIMP - 1 , MIC - 1 /GDF15 , RGS6 , LGRS, PGI/ II , CA242 , dtrmwxhs - 12 , CNX -774 , entrectinib , nilotinib , 1 - (( 3S ,4R ) EN2, UCP2 , a HER - 2 peptide , MUClm , HNP1-3 , L - gluta 4-( 3 -fluorophenyl ) -1- ( 2 -methoxyethylpyrrolidin - 3 -yl ) -3 mine L - tryptophan ( IM862 ) , CPAA - 783 -EPPT1 , serum (4 -methyl -3- (2 -methylpyrimidin - 5 -yl ) -1 - phenyl - 1H -pyra C -peptide , WT1, KIF20A , GV1001, LY6K -177 , PAP- 114 zol- 5 - yl) urea , AG 879, AR - 772 , AR -786 , AR -256 , AR -618 , 128 , E75 , SU18 , SU22 , ANP, TCP - 1 , F56 , WT1, AZ -23 , AZ623 , DS -6051 , Go 6976 , GNF - 5837 , GTX - 186 , TERT572Y , disruptin , TREM - 1 , LFC131 , BPP, TH10 , GW 441756 , LOXO - 101, LOXO - 195 , MGCD516 , BC71 , RC -3095 , RC - 3940 - II , RC -3950 , (KLAKLAK ) 2 , PLX7486 , RXDX101, TPX -0005 , TSR -011 , RGD-( KLAKLAK )2 , NGR-(KLAKLAK ) 2, and SAH -8 ( ABT- 199 , RG7601 , GDC -0199 ), navitoclax (ABT - 263) , ( stapled ). ABT- 737 , TW - 37 , sabutoclax , obatoclax , BIX - 01294 (BIX ), [0216 ] In various methods described herein , platelets are UNC0638 , A - 366 , UNC0642 , DCG066 , UNC0321 , BRD loaded with one or more any of a variety of drugs . In some 4770 , UNC 0224 , UNC 0646 , UNC0631 , BIX -01338 , embodiments , platelets are loaded with a non -miRNA INNO -406 , KX2-391, saracatinib , PP1, PP2 , ruxolitinib , nucleic acid , a non - siRNA nucleic acid , and a non -mRNA lestaurtinib (CEP - 701) , momelotinib (GS -0387 , CYT- 387 ) , nucleic acid ( e.g., non -miRNA , DNA , other naturally or pacritinib (SB1518 ), fedratinib (SAR302503 ) , B12536 , non -naturally occurring nucleic acids, and polymers BI6727 , GSK461364 , , , , car thereof ), including various modifications thereof) . For boplatin , , , , cyclophos example , platelets can be loaded with one or more of phamide , , , , , SPC2996 , SIRNAPLUS, ALN -HTT , ISIS - 199044 , custirsen , doxorubicin , , , fiudara (OGX -011 , ISI- 112989, TV - 1011 ), ISIS - AR - 2.5RX ( ISIS bine , , , , , ARRX, AZD -5312 , ISIS - AZ1Rx , ISIS -560131 ) , ISIS hydroxyurea , , , , , STAT3-2.5RX ( ISIS - STAT3-2.5RX, ISIS -481464 , AZD mechlorethamine , , , methotrxate , 9150 ), BP - 100-1.01 , NOX -A12 (olaptesed peqol) , PNT mitomycin , , , paclitaxel , pem 2258 , ATL - 1103 , RX - 0201, ACT- GRO - 777 , litenimod , etrexed , , streptozocin , tafluposide , temozolo trabedersen ( AP - 2009 ) , IMO - 2055 , OHR -118 , imetelstat , mide , , , , , valru GNKG - 168 , RG -6061 , SPC - 3042 , STAT3 decoys , an anti bicin , , , , and . CD22 antibody -MXD3 antisense oligonucleotide conjugate , [0214 ] In various methods described herein , platelets are AST -008 , ASncmtRNA , an EGFRAS GPNA , ASPH -1047 loaded with one or more any of a variety of drugs. In some (ASPH -0047 ) , STICKY SIRNA , aganirsen , B0-110 , NOX embodiments , platelets are loaded with a protein ( e.g. , an 593 , Adva -R46 , EZN - 4482 , EZN - 4496 , EZN - 3889 , EZN antibody or antibody conjugate ). For example, platelets can 3892 , EZN -4150 , IMO -2125 (HYB -2125 ) , OGX -225 , ATL be loaded with one or more of cetuximab ( ERBITUX® ) , 1101, aqatolimod , AGX - 1053 , AEG - 35156 , qataparsen , necitumumab (PORTRAZZATM , IMC - 11F8 ) , panitumumab ISS - 1018, CpG - 1826 (ODN - 1826 ), CpG -2216 (ODN ( ABX - EGF, VECTIBIX® ), matuzumab ( EMD - 7200 ), 2216 ), CpG -2395 , , pbi- shRNAK - ras LP, LNA nimotuzumab ( h - R3 , BIOMAb EGFR® ) , zalutumab , anti -miR - 155 , ISIS - 20408 , ISIS - 199044 , AP - 11014 , NOX MDX447 , OTSA 101, OTSA101 -DTPA - 90Y, ABBV - 399 , A50 , beclanorsen , ISIS -345794 , ISIS - 15421, GRO -29A , depatuxizumab (humanized mAb 806 , ABT- 806 ), depatuxi LOR -2501 GTI( - 2501) , ISIS - 7597 , ISIS - 3466 , ISIS - 2503 , zumab mafodotin ( ABT -414 ), SAIT301, Sym004 , MAD and GEM -231 . 425 , Modotuximab ( TAB -H49 ) , futuximab (992 DS ) , zalu [0217 ] In various methods described herein , platelets are tumumab , Sym013 , AMG 595 , JNJ- 61186372 , LY3164530 , loaded with one or more any of a variety of drugs. In some IMGN289 , KL - 140 , RO5083945 , SCT200 , CPGJ602 , embodiments , platelets are loaded with an aptamer. For GP369 , BAY1187982 , FPA144 (bemarituzumab ), bevaci example , platelets can be loaded with one or more of zumab ( AVASTIN® ), ranibizumab , trastuzumab (HERCEP ARC126 (RNA ) , AX102 (RNA ) , SL ( 2 )-B (DNA ) , RNV66 TIN® ), pertuzumab (PERJETA? ), trastuzumab - dkst (DNA ), AS1411 (DNA ), FCL - II (DNA , modified form US 2020/0206143 A1 Jul . 2 , 2020 12

AS1411 ) , NOX - A12 (RNA ) , E0727 (RNA ) , CL428 (RNA ) , CH2-) ] , a disulfide - bridged conjugation with synthetic aro KDI130 (RNA ), TuTu2231 (RNA ) , Trimeric apt (DNA ), matics ( see e.g., Chen et al. Org Biomol Chem . 2017 , PNDA -3 (DNA ), TTA140,41 (DNA ), GBI- 1042 (DNA ), 15( 8 ): 1921-1929, which is incorporated by reference herein NAS - 24 ( DNA ) , YJ- 1 (RNA ) , AGE -apt (DNA ) , A - P50 in its entirety ) , blocking N - or C - terminal ends of the peptide (RNA ) , GL21.T (RNA ) , OPN - R3 (RNA ), AGCO3 (DNA ), ( e.g., by N - acylation , N -pyroglutamate , or C - amidation or cy - apt (DNA ), BC15 (DNA ), A9g (RNA ) , ESTA (DNA ) , the addition of carbohydrate chains through , for example , M12-23 (RNA ) , OX40 -apt (RNA ) , De160 (RNA ), PSMA glycosylation with glucose , xylose , hexose ) , an N - terminal 4-1BB -apt (RNA ), CD16a /c -Met -apt (RNA ), VEGF - 4-1BB esterification (phosphoester ), a pegylation modification , and apt (DNA ) , MP7 (DNA ) , aptPD -L1 (DNA ) , R5A1 (RNA ), a reagent or reagents ( see , e.g. , US Publication Application CL -42 ( RNA ) , CD44 - EpCAM aptamer (RNA ) , TIM3 Apt No. 2017/0198335 ) . See.e.g., Vlieghe et al. Drug Discovery ( RNA ) , CD40apt (RNA ) , AptCTLA - 4 (DNA ) , AON -D211 Today , 2010 , 15 , 40-56 , which is incorporated by reference Aptamer (RNA / DNA ) , and BN - 210 . herein in its entirety. [ 0218 ] In some embodiments, a drug loaded into platelets [0219 ] In some embodiments , a drug loaded into platelets is modified . For example , a drug can be modified to increase is modified to include an imaging agent. For example , a drug its stability during the platelet loading process, while the can be modified with an imaging agent in order to image the drug is loaded into the platelet, and /or after the drug's drug loaded platelet in vivo . In some embodiments , a drug release from a platelet . In some embodiments , themodified can be modified with two or more imaging agents (e.g. , any drug's stability is increased with little or no adverse effect on two or more of the imaging agents described herein ). In its activity . For example , themodified drug can have at least some embodiments , a drug loaded into platelets is modified 70 % , 75 % , 80 % , 85 % , 90 % , 95 % , 96 % , 97 % , 98 % , 99 % or with a radioactive metal ion , a paramagnetic metal ion , a more of the activity of the corresponding unmodified drug . gamma - emitting radioactive halogen , a positron - emitting In some embodiments , the modified drug has 100 % ( or radioactive non -metal , a hyperpolarized NMR -active more ) of the activity of the corresponding unmodified drug . nucleus, a reporter suitable for in vivo optical imaging , or a Various modifications that stabilize drugs are known in the beta - emitter suitable for intravascular detection . For art . In some embodiments , the drug is a nucleic acid , which example , a radioactive metal ion can include, but is not nucleic acid is stabilized by one or more of a stabilizing limited to , positron emitters such as 54 Cu , 48V, 52Fe, 55Co , oligonucleotide ( see , e.g., U.S. Application Publication No. 94TC or 68Ga; or gamma- emitters such as 171Tc , 111In , 113 In 2018/0311176 ) , a backbone / side chain modification ( e.g., a or 67Ga . For example , a paramagnetic metal ion can include , 2 - sugar modification such as a 2 '- fluor, methoxy, or amine but is not limited to Gd ( III ), a Mn( II ), a Cu ( II ), a Cr( III ), a substitution , or a 2' - thio ( SH ), 2' - azido ( N3 ) , or 2 '- hy Fe ( III) , a Co (II ) , a Er( II ) , a Ni( II ) , a Eu (III ) or a Dy ( III ) , an droxymethyl ( CH2OH ) modification ), an unnatural element comprising an Fe element, a neodymium iron oxide nucleic acid substitution ( e.g. , an S -glycerol , cyclohexenyl, (NdFeO3 ) or a dysprosium iron oxide (DyFeO3 ) . For and / or threose nucleic acid substitution , an L -nucleic acid example , a paramagnetic metal ion can be chelated to a substitution , a locked nucleic acid ( LNA ) modification ( e.g. , polypeptide or a monocrystalline nanoparticle . For example, the ribose moiety of an LNA nucleotide is modified with an a gamma- emitting radioactive halogen can include , but is extra bridge connecting the 2 ' oxygen and 4 carbon ), not limited to 1231, 1311 or 77Br. For example , a positron conjugation with PEG , a nucleic acid bond modification or emitting radioactive non -metal can include , but is not lim replacement ( e.g. , a phosphorothioate bond , a methylphos ited to 11C , 13N , 150 , 177 , 18F, 75Br, 76Br or 1241. For phonate bond, or a phosphorodiamidate bond ) , a reagent or example , a hyperpolarized NMR - active nucleus can include, reagents ( e.g., intercalating agents such as coralyne, neo but is not limited to 13C , 15N , 1 ° F , 2ºSi and 31P . For example , mycin , and ellipticine ; also see US Publication Application a reporter suitable for in vivo optical imaging can include , Nos. 2018/0312903 and 2017/0198335 , each of which are but is not limited to any moiety capable of detection either incorporated herein by reference in their entireties , for directly or indirectly in an optical imaging procedure . For further examples of stabilizing reagents ). In some embodi example , the reporter suitable for in vivo optical imaging ments , the drug is a polypeptide, which polypeptide is can be a light scatterer ( e.g., a colored or uncolored particle ), stabilized by one or more of cyclization of the peptide a light absorber or a light emitter . For example , the reporter sequence [ e.g., between side chains or ends of the peptide can be any reporter that interacts with light in the electro sequence ( for example , head to tail, N -backbone to N -back magnetic spectrum with wavelengths from the ultraviolet to bone , end to N - backbone , end to side chain , side chain to the near infrared . For example , organic chromophoric and N - backbone , side chain to side chain ) through disulfide , fluorophoric reporters include groups having an extensive lanthionine , dicarba, , or lactam bridges ], a back delocalized electron system , e.g. cyanines, merocyanines , bone / side chain modification , an unnatural residue substitu indocyanines, phthalocyanines, naphthalocyanines, triph tion ( e.g., a D - amino acid , an N -methyl - a -amino acid , a enylmethines, porphyrins, pyrilium dyes, thiapyrilium dyes , non - proteogenic constrained amino acid or a ß -amino acid ), squarylium dyes , croconium dyes , azulenium dyes, indoa a peptide bond modification or replacement [ e.g., NH -amide nilines , benzophenoxazinium dyes , benzothiaphenothiaz alkylation , the carbonyl function of the peptide bond can be inium dyes, , napthoquinones, indathrenes , replaced by CH2 (reduced bond : CH2- NH— ) , CGS) phthaloylacridones , trisphenoquinones , azo dyes , intramo ( endothiopeptide , CES) -NH- ) or PO2H ( phosphona lecular and intermolecular charge - transfer dyes and dye mide, —P ( = O )OH - NH— ) , the NH - amide bond can be complexes , tropones , tetrazines, b /s ( dithiolene ) complexes, exchanged by O (depsipeptide , CO - 0 ) , S ( thioester, bus (benzene - dithiolate ) complexes, iodoaniline dyes , b /sts . -C0 - S_ ) or CH2 (ketomethylene , CO CH2- ), a O -dithiolene ) complexes. For example , the reporter can be, retro - inverso bond ( NH4C0— ), a methylene - oxy bond but is not limited to a fluorescent, a bioluminescent, or CH2- ) , a thiomethylene bond ( CH2- S ) , a carba bond chemiluminescent polypeptide. For example , a fluorescent CH2- CH2- ), and a hydroxyethylene bond ( CHOH or chemiluminescent polypeptide is a green florescent pro US 2020/0206143 A1 Jul . 2 , 2020 13

tein (GFP ), a modified GFP to have different absorption / buffer comprising DMSO and comprising a salt, a base , a emission properties , a luciferase , an aequorin , an obelin , a loading agent, and optionally ethanol, to form the cargo mnemiopsin , a berovin , or a phenanthridinium ester . For loaded platelets. example , a reporter can be , but is not limited to rare earth [0224 ] In some embodiments the loading buffer , and/ or the metals ( e.g. , europium , samarium , terbium , or dysprosium ), liquid medium , may comprise one or more salts selected or fluorescent nanocrystals ( e.g., quantum dots ) . For from phosphate salts , sodium salts , potassium salts , calcium example , a reporter may be a chromophore that can include, salts , magnesium salts , and any other salt that can be found but is not limited to fluorescein , sulforhodamine 101 ( Texas in blood or blood products , or that is known to be useful in Red ), rhodamine B , rhodamine 6G , rhodamine 19 , indocya drying platelets , or any combination of two or more of these. nine green, Cy2 , Cy3 , Cy3.5 , Cy5, Cy5.5 , Cy7 , Marina [ 0225 ] Preferably , these salts are present in the composi Blue, Pacific Blue , Oregon Green 88 , Oregon Green 514 , tion at an amount that is about the same as is found in whole tetramethylrhodamine , and Alexa Fluor 350 , Alexa Fluor blood . 430 , Alexa Fluor 532 , Alexa Fluor 546 , Alexa Fluor 555 , Alexa Fluor 568, Alexa Fluor 594 , Alexa Fluor 633 , Alexa [ 0226 ] In some embodiments , the drug - loaded platelets Fluor 647 , Alexa Fluor 660 , Alexa Fluor 680 , Alexa Fluor are prepared by incubating the platelets with the drug in the 700 , and Alexa Fluor 750. For example , a beta -emitter can liquid medium for different durations at or at different include, but is not limited to radio metals 67Cu , 89Sr, 9° Y, temperatures from 15-45 ° C., or about 37 ° C. (cell to drug 153Sm , 185Re, 188Re or 1921r, and non -metals 32P, 33P, 38S , volume ratio of 1 : 2 ). 38C1, 39C1, 82Br and 83Br. In some embodiments , a drug [0227 ] In some embodiments , the platelets form a suspen loaded into platelets can be associated with gold or other sion in a liquid medium at a concentration from 10,000 equivalent metal particles ( such as nanoparticles ). For platelets/ uL to 10,000,000 platelets/ ul , such as 50,000 example , a metal particle system can include, but is not platelets/ uL to 2,000,000 platelets/ uL , such as 100,000 limited to gold nanoparticles ( e.g. , NanogoldTM ) . platelets /uL to 500,000 platelets /uL , such as 150,000 plate [0220 ] In some embodiments , a drug loaded into platelets lets/ uL to 300,000 platelets/ ?L , such as 200,000 platelets / that is modified with an imaging agent is imaged using an UL . imaging unit. The imaging unit can be configured to image [0228 ] In some embodiments , one or more other compo the drug loaded platelets in vivo based on an expected nents may be loaded in the platelets . In some embodiments , property ( e.g., optical property from the imaging agent) to be the one or more other components may be loaded concur characterized . For example, imaging techniques ( in vivo rently with the drug. In some embodiments, the one or more other components and the drug may be loaded sequentially imaging using an imaging unit ) that can be used , but are not in either order . Components may include an agent ( e.g. , an limited to are : computer assisted tomography (CAT ) , mag anti- aggregation agent ) that reduces or prevents platelet netic resonance spectroscopy (MRS ) , magnetic resonance aggregation and activation during the loading process . imaging (MRI ) , positron emission tomography (PET ) , Exemplary components ( e.g. , anti- aggregation agents ) may single -photon emission computed tomography (SPECT ) , or include an anti- aggregation agent such as, Prostaglandin E1 bioluminescence imaging (BLI ) . Chen Z., et . al. , Advance of or Prostacyclin and or EDTA / EGTA to prevent platelet Molecular Imaging Technology and Targeted Imaging Agent aggregation and activation during the loading process. Addi in Imaging and Therapy, Biomed Res Int. , February 13 , doi: tional non - limiting anti- aggregation agents may include, 10.1155/ 2014 /819324 ( 2014 ) have described various imag GR144053 , FR171113, aspirin , MeSADP , PSB 0739 , Can ing techniques and which is incorporated by reference herein grelor, Tirofiban ( e.g., AggrastatTM ) , and Mito TEMPO , in its entirety . N - acetyyl- L - cysteine , cytcochalasin D , Staurosporine , [ 0221 ] In some embodiments , such as embodiments Mepacrine, actezolamide , or dichloroacetate . These compo wherein the platelets are treated with the drug and the buffer nents may be used alone or in combination with one another . sequentially as disclosed herein , the drug may be loaded in [0229 ] Accordingly , in some embodiments , an agent suit a liquid medium that may be modified to change the pro able for treatment of cancer , such as doxorubicin , may be portion ofmedia components or to exchange components for loaded together with , prior to , or following , a GPIIB / IIIa similar products , or to add components necessary for a given inhibitor. In some embodiments , the cancer is acute lym application . phoblastic leukemia . In some embodiments , the cancer is [0222 ] In some embodiments the loading buffer , and / or the acute myeloid leukemia . In some embodiments , the cancer liquid medium , may comprise one or more of a ) water or a is breast cancer or metastasized breast cancer. In some saline solution , b ) one or more additional salts , or c ) a base . embodiments , the cancer is gastric cancer. In some embodi In some embodiments , the loading buffer , and / or the liquid ments , the cancer is Hodgkin lymphoma. In some embodi medium , may comprise one or more of a ) DMSO , b ) one or ments , the cancer is neuroblastoma. In some embodiments , more salts , or c ) a base . the cancer is Non -Hodgkin lymphoma. In some embodi [0223 ] In some embodiments the loading agent is loaded ments , the cancer is ovarian cancer . In some embodiments , into the platelets in the presence of an aqueous medium . In the cancer is small cell lung cancer . In some embodiments , some embodiments the loading agent is loaded in the the cancer is soft tissue and bone sarcomas. In some embodi presence of a medium comprising DMSO . As an example , ments , the cancer is thyroid cancer. In some embodiments , one embodiment of the methods herein comprises treating the cancer is transitional cell bladder cancer. In some platelets with a drug and with an aqueous loading buffer embodiments , the cancer is Wilms tumor. comprising a salt , a base , a loading agent, and optionally at [0230 ] Accordingly , in some embodiments, an agent suit least one organic solvent, to form the cargo -loaded platelets . able for treatment of cancer , such as olaparib (also known as As an example , one embodiment of the methods herein AZD - 2281 , MK -7339 , trade name Lynparza® ), may be comprises treating platelets with a drug and with a loading loaded together with , prior to , or following , a GPIIb / IIIa US 2020/0206143 A1 Jul . 2 , 2020 14

inhibitor. In some embodiments , the cancer is ovarian can [0238 ] In some embodiments , the one or more other cer. In some embodiments , the cancer is breast cancer. components that are loaded in the platelets do not comprise [0231 ] Accordingly, in some embodiments , an agent suit EDTA . able for treatment of cancer, such as paclitaxel ( Taxol® ) , [0239 ] The table below shows the effect of the addition of may be loaded together with , prior to , or following , a antiplatelet compounds on DOX - induced platelet aggrega GPIIb / IIIa inhibitor. In some embodiments , the cancer is tion : TABLE A Max Platelet Count Recom (Normalized to Untreated ) mended 0.5 0.6 1.2 concen mg/mL mg/mL mM mM # Reagent Target Source tration DOX DOX DOX DOX GR144053 GPIIB / IIIa PMID : IC50 37 nM 83 % 42 % N / A N / A inhibitor 9700979 2 PGE1 P2Y1 PMID : 22 nM - 1 uM N / A N / A N / A N / A receptor 22385219 activation PMID : Inhibitor 22268418 3 MeSADP P2Y1 , PMID : 10 UM 33 % 10 % N / A N / A P2Y12 , and 9442039 P2Y13 Agonist 4 FR171113 PAR1 PMID : 0.3 UM 51 % 16 % N / A N / A antagonist 10611442 5 Aspirin COX PMID : 40-500 UM 52 % 15 % N / A N / A ( ASA ) inhibitor 3370916 6 Cangrelor P2Y12 PMID : 1 UM 47 % 26 % N / A N / A Inhibitor 23236426 7 PSB 0739 P2Y12 PMID : 500 nM 26 % 18 % N / A N / A Inhibitor 27695417 8 N Thiol PMID : 5 mM N / A N / A 73 % N / A Acetyl Supplement 194 282 cysteine page 1179 9 Mito TEMPO ROS PMID : 10 ?M N / A N / A N / A 37 % Antagonist 25988386 methods 4.3 10 Tirofiban GPIIb / IIIa PMID : 5 uM N / A N / A 73 % N / A inhibitor 11406724 abstract

Kaposi sarcoma. In some embodiments , the cancer is breast [0240 ] The table below shows alternatively proposed anti cancer. In some embodiments, the cancer is non - small cell platelet compounds to combat DOX - induced platelet aggre lung cancer . In some embodiments , the cancer is ovarian gation : cancer . [ 0232 ] In some embodiments , an agent suitable for treat TABLE B mentof cancer, such as doxorubicin , may be loaded together with , prior to , or following , P2Y1 receptor activation inhibi Recommended tor, a P2Y1 agonist, P2Y12 agonist, a P2Y13 agonist , a PAR # Reagent Target Source concentration 1 antagonist, a COX inhibitor, a P2Y12 inhibitor , a thiol 1 Cytochalasin actin PMID : 10682859 10 UM supplement, a ROS antagonist , an actin polymerization D polymerization page 357 2 Stauro protein kinase PMID : 10051374 methods 25 nM to inhibitor, protein kinase C inhibitor , phospholipase A2 sporine C inhibitor “ PKC studies " 10 UM inhibitor , Rho kinase inhibitor, a carbonic anhydrase inhibi PMID : 11895774 tor, or a PDK inhibitor. ( function or reagent ) 2.5-20 UM [0233 ] In some embodiments , the one or more other 3 Mepacrine Phospholipase PMID : 3931692 components that are loaded in the platelets comprise Pros A2 inhibitor taglandin E1 (PGE1 ) or Prostacyclin . [0234 ] In some embodiments , the one or more other [0241 ] In some embodiments , other components may components that are loaded in the platelets do not comprise include imaging agents . For example , an imaging agent can Prostaglandin El or Prostacyclin . include , but is not limited to a radioactive metal ion , a [0235 ] In some embodiments , the one or more other paramagnetic metal ion , a gamma - emitting radioactive halo components that are loaded in the platelets comprise EGTA . gen , a positron - emitting radioactive non -metal , a hyperpo [ 0236 ] In some embodiments , the one or more other larized NMR - active nucleus, a reporter suitable for in vivo components that are loaded in the platelets do not comprise optical imaging , or a beta -emitter suitable for intravascular EGTA . detection . For example , a radioactive metal ion can include , [0237 ] In some embodiments , the one or more other but is not limited to , positron emitters such as 54Cu , 48V, components that are loaded in the platelets comprise EDTA . 52Fe , 55Co , 94Tc or 68Ga; or gamma- emitters such as US 2020/0206143 A1 Jul . 2 , 2020 15

171Tc , 111In , 113In , or 67Ga. For example , a paramagnetic the drug loaded platelets in vivo based on an expected metal ion can include , but is not limited to Gd (III ) , a Mn ( II ) , property (e.g. , optical property from the imaging agent) to be a Cu ( II ), a Cr ( III ), a Fe ( III ), a Co ( II ), a Er( II ), a Ni( II) , a characterized . For example, imaging techniques (in vivo Eu ( III ) or a Dy( III ) , an element comprising an Fe element, imaging using an imaging unit ) that can be used , but are not a neodymium iron oxide (NdFeO3 ) or a dysprosium iron limited to are : computer assisted tomography (CAT ) , mag oxide (DyFeO3 ) . For example , a paramagnetic metal ion can netic resonance spectroscopy (MRS ), magnetic resonance be chelated to a polypeptide or a monocrystalline nanopar imaging (MM ) , positron emission tomography (PET ) , ticle . For example, a gamma- emitting radioactive halogen single -photon emission computed tomography (SPECT ) , or can include, but is not limited to 1231, 1311 or 77Br. For bioluminescence imaging (BLI ) . Chen Z. et. al ., ( 2014 ) have example , a positron - emitting radioactive non -metal can described various imaging techniques and which is incor include, but is not limited to 11C , 13N , 150 , 17F , 18F , 75Br, porated by reference herein in its entirety . 76Br or 1241. For example , a hyperpolarized NMR -active [0243 ] In some embodiments , the drug -loaded platelets nucleus can include , but is not limited to 13C , 15N , 19F , are prepared by incubating the platelets with the drug in the 29Si and 31P . For example , a reporter suitable for in vivo liquid medium for different durations. The step of incubating optical imaging can include, but is not limited to any moiety the platelets to load one or more cargo , such as a drug ( s ) , capable of detection either directly or indirectly in an optical includes incubating the platelets for a time suitable for imaging procedure . For example , the reporter suitable for in loading , as long as the time, taken in conjunction with the vivo optical imaging can be a light scatterer ( e.g., a colored temperature, is sufficient for the drug to come into contact or uncolored particle ) , a light absorber or a light emitter. For with the platelets and , preferably , be incorporated , at least to example , the reporter can be any reporter that interacts with some extent, into the platelets. For example , in some light in the electromagnetic spectrum with wavelengths from embodiments , the drug - loaded platelets are prepared by the ultraviolet to the near infrared . For example , organic incubating the platelets with the drug in the liquid medium chromophoric and fluorophoric reporters include groups for at least about 5 minutes (mins ) ( e.g., at least about 20 having an extensive delocalized electron system , e.g. cya mins , about 30 mins, about 1 hour (hr ) , about 2 hrs , about 3 nines, merocyanines, indocyanines, phthalocyanines , naph hrs, about 4 hrs, about 5 hrs , about 6 hrs , about 7 hrs , about thalocyanines , triphenylmethines , porphyrins, pyrilium 8 hrs, about 9 hrs , about 10 hrs , about 12 hrs , about 16 hrs, dyes, thiapyrilium dyes , squarylium dyes, croconium dyes , about 20 hrs , about 24 hrs , about 30 hrs, about 36 hrs , about azulenium dyes, indoanilines, benzophenoxazinium dyes , 42 hrs , about 48 hrs , or at least about 48 hrs. In some benzothiaphenothiazinium dyes , anthraquinones , napthoqui embodiments , the drug - loaded platelets are prepared by nones , indathrenes, phthaloylacridones , trisphenoquinones, incubating the platelets with the drug in the liquid medium azo dyes , intramolecular and intermolecular charge- transfer for no more than about 48 hrs ( e.g., no more than about 20 dyes and dye complexes , tropones , tetrazines , b / s ( dithi mins , about 30 mins, about 1 hour (hr ) , about 2 hrs , about 3 olene ) complexes, bus (benzene -dithiolate ) complexes , hrs , about 4 hrs , about 5 hrs , about 6 hrs, about 7 hrs, about iodoaniline dyes, b / stS.O -dithiolene ) complexes. For 8 hrs , about 9 hrs , about 10 hrs , about 12 hrs, about 16 hrs , example , the reporter can be, but is not limited to a fluo about 20 hrs , about 24 hrs, about 30 hrs , about 36 hrs , or no rescent, a bioluminescent, or chemiluminescent polypeptide . more than about 42 hrs ) . In some embodiments , the drug For example , a fluorescent or chemiluminescent polypeptide loaded platelets are prepared by incubating the platelets with is a green florescent protein (GFP ) , a modified GFP to have the drug in the liquid medium from about 10 mins to about different absorption / emission properties, a luciferase , an 48 hours (e.g. , from about 20 mins to about 36 hrs , from aequorin , an obelin , a mnemiopsin , a berovin , or a phenan about 30 mins to about 24 hrs, from about 1 hr to about 20 thridinium ester . For example , a reporter can be , but is not hrs , from about 2 hrs to about 16 hours, from about 10 mins limited to rare earth metals (e.g. , europium , samarium , to about 24 hours , from about 20 mins to about 12 hours , terbium , or dysprosium ), or fluorescent nanocrystals ( e.g., from about 30 mins to about 10 hrs, or from about 1 hr to quantum dots ). For example , a reporter may be a chro about 6 hrs . In one embodiment, treating platelets , platelet mophore that can include , but is not limited to fluorescein , derivatives, or thrombosomes with a drug, a liquid medium , sulforhodamine 101 ( Texas Red ), rhodamine B , rhodamine a buffer comprising a salt, a base , a loading agent, and 6G , rhodamine 19, indocyanine green , Cy2 , Cy3 , Cy3.5 , optionally at least one organic solvent, and / or with any Cy5 , Cy5.5 , Cy7 , Marina Blue, Pacific Blue, Oregon Green loading protocol described herein to form the drug - loaded 88 , Oregon Green 514 , tetramethylrhodamine , and Alexa platelets comprises contacting the platelets, platelet deriva Fluor 350 , Alexa Fluor 430 , Alexa Fluor 532 , Alexa Fluor tives , or thrombosomes with a drug , a liquid medium , a 546 , Alexa Fluor 555 , Alexa Fluor 568, Alexa Fluor 594 , buffer comprising a salt, a base, a loading agent, and Alexa Fluor 633 , Alexa Fluor 647 , Alexa Fluor 660, Alexa optionally at least one organic solvent and / or with any Fluor 680 , Alexa Fluor 700 , and Alexa Fluor 750. For loading protocol described herein for a period of time, such example , a beta - emitter can include, but is not limited to as a period of 5 minutes to 48 hours , such as 2 hours . radio metals 67Cu, 89Sr , 90Y, 153 Sm , 185Re , 188Re or [0244 ] In some embodiments , the drug- loaded platelets 1921r , and non -metals 32P , 33P , 385 , 38C1, 39C1, 82Br and are prepared by incubating the platelets with the drug in the 83Br. In some embodiments , a drug loaded into platelets can liquid medium at different temperatures . The step of incu be associated with gold or other equivalent metal particles bating the platelets to load one or more cargo , such as a ( such as nanoparticles ) . For example , a metal particle system drug ( s) , includes incubating the platelets with the drug in the can include , but is not limited to gold nanoparticles (e.g. , liquid medium at a temperature that, when selected in NanogoldTM ). conjunction with the amount of time allotted for loading , is [ 0242 ] In some embodiments , the one or more imaging suitable for loading. In general, the platelets with the drug in agents loaded concurrently with a drug is imaged using an the liquid medium are incubated at a suitable temperature imaging unit. The imaging unit can be configured to image ( e.g., a temperature above freezing ) for at least a sufficient US 2020/0206143 A1 Jul. 2 , 2020 16 time for the drug to come into contact with the platelets . In about 40 mM , from about 8 mM to about 30 mM , about 10 embodiments , incubation is conducted at 37 ° C. In certain mM to about 25 mM ) about of the one or more buffers . In embodiments , incubation is performed at 4 ° C. to 45 ° C., some embodiments , the loading buffer includes about 10 such as 15º C. to 42 ° C. For example , in embodiments , mM , about 20 mM , about 25 mM , or about 30 mM of the incubation is performed at 35 ° C. to 40 ° C. ( e.g., 37 ° C.) for one or more buffers . 110 to 130 ( e.g., 120 ) minutes and for as long as 24-48 [0250 ] In some embodiments, the loading buffer includes hours . one or more saccharides , such as monosaccharides and [0245 ] In some embodiments of a method of preparing disaccharides, including sucrose , maltose , trehalose , glu drug - loaded platelets disclosed herein , the method further cose, mannose , dextrose, and xylose . In some embodiments , comprises acidifying the platelets , or pooled platelets , to a the loading buffer includes from about 10 mM to about pH of about 6.0 to about 7.4 , prior to a treating step disclosed 1,000 mM of the one or more saccharides. In some embodi herein . In some embodiments , the method comprises acidi ments , the loading buffer includes from about 50 to about fying the platelets to a pH ofabout 6.5 to about 6.9 . In some 500 mM of the one or more saccharides. In embodiments , embodiments , the method comprises acidifying the platelets one or more saccharides is present in an amount of from 10 to a pH of about 6.6 to about6.8 . In some embodiments , the mM 10 to 500 mM . In some embodiments, one or more acidifying comprises adding to the pooled platelets a solu saccharides is present in an amount of from 50 mM to 200 tion comprising Acid Citrate Dextrose . mM . In embodiments , one ormore saccharides is present in [ 0246 ] In some embodiments , the platelets are isolated an amount from 100 mM to 150 mM . prior to a treating step . In some embodiments , the method [0251 ] In some embodiments , the loading buffer includes further comprises isolating platelets by using centrifugation . adding an organic solvent, such as ethanol , to the loading In some embodiments , the centrifugation occurs at a relative solution . In such a loading buffer , the solvent can range from centrifugal force (RCF ) of about 800 g to about 2000 g . In about 0.1 % (v /v ) to about 5.0 % ( v /v ) , such as from about some embodiments , the centrifugation occurs at relative 0.3 % (v / v ) to about 3.0 % (v /v ) , or from about 0.5 % ( v / v ) to centrifugal force (RCF ) of about 1300 g to about 1800 g . In about 2 % ( v/ v ). some embodiments , the centrifugation occurs at relative [0252 ] In some embodiments , the drug comprises doxo centrifugal force (RCF ) of about 1500 g . In some embodi rubicin (“ DOX ” ) . DOX interacts with DNA by intercalation ments , the centrifugation occurs for about 1 minute to about and inhibits macromolecular biosynthesis ( Tacar, O. et. al. , 60 minutes . In some embodiments , the centrifugation occurs Doxorubicin : an update on anticancer molecular action , for about 10 minutes to about 30 minutes . In some embodi toxicity and novel drug delivery systems, The Journal of ments , the centrifugation occurs for about 30 minutes. Pharmacy and Pharmacology , 65 ( 2 ) : 157-70 . doi: 10.1111 / [0247 ] In some embodiments , the platelets are at a con j.2042-7158.2012.01567.x . PMID 23278683 ( 2013 ), which centration from about 2,000 platelets/ ul to about 500,000 , is incorporated herein by reference ) . 000 platelets /ul . In some embodiments , the platelets are at a [0253 ] In some embodiment, the drug comprises pacli concentration from about 50,000 platelets /u1 to about 4,000 , taxel . In some embodiment, the drug comprises paclitaxel 000 platelets /ul . In some embodiments , the platelets are at that is not in the presence of Cremophor EL . In some a concentration from about 100,000 platelets / ul to about embodiments , the drug comprises paclitaxel that is not in the 300,000,000 platelets / ul. In some embodiments , the platelets presence ( e.g., an excipient ) of a polyexthoxylated castor oil. are at a concentration from about 1,000,000 to about 2,000 , For example , paclitaxel that is not in the presence of an 000. In some embodiments , the platelets are at a concentra excipient comprising a polyethylene glycol ether. tion of about 200,000,000 platelets/ ul . [0254 ] In some embodiments , the drug comprises a poly [0248 ] In some embodiments , the buffer is a loading buffer ADP ribose polymerase (PARP ) inhibitor (PARPi ) . PARPis comprising the components as listed in Table 1 herein . In prevent the normal repair of DNA breaks which in turn leads some embodiments , the loading buffer comprises one or to cell death . In some embodiments , the PARPi is olaparib . more salts , such as phosphate salts , sodium salts , potassium [0255 ] In some embodiments , the method further com salts , calcium salts , magnesium salts , and any other salt that prises incubating the drug in the presence of the loading can be found in blood or blood products . Exemplary salts buffer prior to the treatment step . In some embodiments , the include sodium chloride (NaCl ) , potassium chloride (KCI ) , method further comprises incubating the loading buffer and and combinations thereof. In some embodiments , the load a solution comprising the drug and water at about 37° C. ing buffer includes from about 0.5 mM to about 100 mM of using different incubation periods. In some embodiments , the one or more salts . In some embodiments , the loading the solution includes a concentration of about 1 nM to about buffer includes from about 1 mM to about 100 mM ( e.g. , 1000 mM of the drug . In some embodiments , the solution about 2 mM to about 90 mM , about 2 mM to about 6 mM , includes a concentration of about 10 nM to about 10 mM of about 50 mM to about 100 mM , about 60 mM to about 90 the drug . In some embodiments , the solution includes a mM , about 70 to about 85 mM ) about of the one or more concentration of about 100 nM to 1 mM of the drug. In some salts . In some embodiments , the loading buffer includes embodiments , the solution includes a concentration of from about 5 mM , about 75 mm , or about 80 mM of the one or about 10 mg/ ml of water to about 100 mg/ml . In some more salts . embodiments , the solution includes a concentration of from [0249 ] In some embodiments , the loading buffer includes about 20 mg/ ml of water to about 80 mg/ml . In some one or more buffers , e.g., N - 2 -hydroxyethylpiperazine -N' embodiments , the solution includes a concentration of from 2 -ethanesulfonic acid (HEPES ) , or sodium -bicarbonate about 40 mg/ ml of water to about 60 mg/ml . In some (NaHCO3 ) . In some embodiments , the loading buffer embodiments , the incubation of the drug in the presence of includes from about 5 to about 100 mM of the one or more the loading buffer is performed from about 1 minute to about buffers. In some embodiments , the loading buffer includes 2 hours. In some embodiments , the incubation is performed from about 5 to about 50 mM (e.g. , from about 5 mM to at an incubation period of from about 5 minutes to about 1 US 2020/0206143 A1 Jul . 2 , 2020 17 hour . In some embodiments , the incubation is performed at preferentially collecting the dried mixture . The dried com an incubation period of from about 10 minutes to about 30 position in some embodiments is stable for at least six minutes . In some embodiments , the incubation is performed months at temperatures that range from -20 ° C. or lower to at an incubation period of about 20 minutes. 90 ° C. or higher. [0256 ] In some embodiments , the method further com prises mixing the platelets and the drug in the presence of the TABLE B loading buffer at 37 ° C., using a platelet to drug volume ratio of 1 : 2 . In some embodiments , the method further comprises Exemplary Lyophilization Protocol incubating the platelets and the drug in the presence of the Temp . Pressure loading buffer at 37 ° C. using a platelet to drug volume ratio Step Set Type Duration Set of 1 :2 , using different incubation periods. In some embodi Freezing Step F1 -50 ° C. Ramp Var N / A ments , the incubation is performed at an incubation period F2 -50 ° C. Hold 3 Hrs N / A of from about 5 minutes to about 12 hours . In some Vacuum Pulldown F3 -50 ° Hold Var N / A embodiments , the incubation is performed at an incubation Primary Dry P1 -40 ° Hold 1.5 Hrs O mT P2 -350 Ramp 2 Hrs O mT period of from about 10 minutes to about 6 hours . In some P3 -250 Ramp 2 Hrs 0 mT embodiments , the incubation is performed at an incubation P4 -17 ° C. Ramp 2 Hrs 0 mT period of from about 15 minutes to about 3 hours . In some P5 0 ° C. Ramp 1.5 Hrs 0 mT embodiments , the incubation is performed at an incubation P6 27 ° C. Ramp 1.5 Hrs 0 mT period of about 2 hours. P7 27 ° C. Hold 16 Hrs O mT [0257 ] In some embodiments , the concentration of drug in Secondary Dry si 27 ° C. Hold > 8 Hrs OmT the drug - loaded platelets is from about 1 nM to about 1000 mM . In some embodiments , the concentration of drug in the [ 0263 ] In some embodiments , the step of drying the drug drug -loaded platelets is from about 10 nM to about 10 mM . loaded platelets that are obtained as disclosed herein , such as In some embodiments , the concentration of drug in the the step of freeze - drying the drug - loaded platelets that are drug- loaded platelets is from about 100 nM to 1 mM . obtained as disclosed herein , comprises incubating the plate [0258 ] In some embodiments , the method further com lets with a lyophilizing agent ( e.g., a non - reducing disac prises drying the drug - loaded platelets . In some embodi charide . Accordingly , in some embodiments , the methods ments , the drying step comprises freeze- drying the drug for preparing drug- loaded platelets further comprise incu loaded platelets . In some embodiments , the method further bating the drug- loaded platelets with a lyophilizing agent comprises rehydrating the drug- loaded platelets obtained [0264 ] In some embodiments the lyophilizing agent is a from the drying step . saccharide . In some embodiments the saccharide is a disac [ 0259 ] In some embodiments , drug - loaded platelets are charide , such as a non -reducing disaccharide ) . prepared by using any one of the methods provided herein . [ 0265 ] In some embodiments , the platelets are incubated [0260 ] In some embodiments , rehydrated drug - loaded with a lyophilizing agent for a sufficient amount of time and platelets are prepared by any one method comprising rehy at a suitable temperature to load the platelets with the drating the drug - loaded platelets provided herein . lyophilizing agent. Non - limiting examples of suitable [0261 ] The drug - loaded platelets may be then used , for lyophilizing agents are saccharides , such as monosaccha example , for therapeutic applications as disclosed herein . As rides and disaccharides, including sucrose, maltose , treha another example , the drug -loaded platelets may be lose, glucose ( e.g., dextrose ), mannose , and xylose . In some employed in functional assays. In some embodiments , the embodiments , non - limiting examples of lyophilizing agent drug - loaded platelets are cold stored , cryopreserved , or include serum albumin , dextran , polyvinyl pyrrolidone lyophilized ( to produce thrombosomes ) prior to use in ( PVP ), starch , and hydroxyethyl starch (HES ) . In some therapy or in functional assays . embodiments , exemplary lyophilizing agents can include a [ 0262] Any known technique for drying platelets can be high molecular weight polymer, into the loading composi used in accordance with the present disclosure , as long as the tion . By “ high molecular weight” it is meant a polymer technique can achieve a final residual moisture content of having an average molecular weight of about or above 70 less than 5 % . Preferably, the technique achieves a final kDa. Non - limiting examples are polymers of sucrose and residual moisture content of less than 2 % , such as 1 % , 0.5 % , epichlorohydrin . In some embodiments , the lyophilizing or 0.1 % . Non - limiting examples of suitable techniques are agent is polysucrose. Although any amount of high molecu freeze -drying (lyophilization ) and spray -drying . A suitable lar weight polymer can be used as a lyophilizing agent, it is lyophilization method is presented in Table A. Additional preferred that an amount be used that achieves a final exemplary lyophilization methods can be found in U.S. Pat. concentration of about 3 % to 10 % (w /v ), such as 3 % to 7 % , Nos. 7,811,558 , 8,486,617 , and 8,097,403 . An exemplary for example 6 % . spray -drying method includes: combining nitrogen , as a [0266 ] In some embodiments , the process for preparing a drying gas, with a loading buffer according to the present composition includes adding an organic solvent, such as disclosure , then introducing the mixture into GEA Mobile ethanol, to the loading solution . In such a loading solution , Minor spray dryer from GEA Processing Engineering , Inc. the solvent can range from 0.1 % to 5.0 % ( v / v ) . ( Columbia Md. , USA ), which has a Two -Fluid Nozzle [ 0267 ] Within the process provided herein for making the configuration , spray drying the mixture at an inlet tempera compositions provided herein , addition of the lyophilizing ture in the range of 150 ° C. to 190 ° C., an outlet temperature agent can be the last step prior to drying . However, in some in the range of 65 ° C. to 100 ° C., an atomic rate in the range embodiments , the lyophilizing agent is added at the same of 0.5 to 2.0 bars , an atomic rate in the range of 5 to 13 kg/ hr, time or before the drug , the cryoprotectant, or other com a nitrogen use in the range of60 to 100 kg /hr , and a run time ponents of the loading composition . In some embodiments , of 10 to 35 minutes. The final step in spray drying is the lyophilizing agent is added to the loading solution , US 2020/0206143 A1 Jul . 2 , 2020 18

thoroughly mixed to form a drying solution , dispensed into to improve pH maintenance of the compositions provided a drying vessel (e.g. , a glass or plastic serum vial, a herein . For example , in some embodiments , the SAN ratio lyophilization bag ) , and subjected to conditions that allow of the container can be at least about 2.0 mL / cm2 ( e.g. , at for drying of the solution to form a dried composition . least about 2.1 mL / cm², at least about 2.2 mL/ cm² , at least [0268 ] An exemplary saccharide for use in the composi about 2.3 mL /cm² , at least about 2.4 mL/ cm², at least about tions disclosed herein is trehalose . Regardless of the identity 2.5 mL / cm², at least about 2.6 mL /cm² , at least about 2.7 of the saccharide, it can be present in the composition in any mL / cm², at least about 2.8 mL/ cm², at least about 2.9 suitable amount. For example , it can be present in an amount mL/ cm² , at least about 3.0 mL / cm², at least about 3.1 of 1 mM to 1 M. In some embodiments , it is present in an mL / cm², at least about 3.2 mL / cm², at least about 3.3 amount of from 10 mM 10 to 500 mM . In some embodi mL / cm², at least about 3.4 mL / cm², at least about 3.5 ments , it is present in an amount of from 20 mM to 200 mM . mL /cm² , at least about 3.6 mL /cm² , at least about 3.7 In some embodiments , it is present in an amount from 40 mL / cm², at least about 3.8 mL/ cm², at least about 3.9 mM to 100 mM . In various embodiments , the saccharide is mL / cm², at least about 4.0 mL / cm², at least about 4.1 present in different specific concentrations within the ranges mL /cm² , at least about 4.2 mL /cm² , at least about 4.3 recited above, and one of skill in the art can immediately mL / cm², at least about 4.4 mL/ cm², at least about 4.5 understand the various concentrations without the need to mL / cm², at least about 4.6 mL / cm², at least about 4.7 specifically recite each herein . Where more than one sac mL / cm², at least about 4.8 mL / cm² , at least about 4.9 charide is present in the composition , each saccharide can be mL / cm², or at least about 5.0 mL / cm². In some embodi present in an amount according to the ranges and particular ments, the SAN ratio of the container can be at most about concentrations recited above . 10.0 mL / cm² ( e.g., at most about 9.9 mL /cm² , at most about [0269 ] The step of incubating the platelets to load them 9.8 mL/ cm², at most about 9.7 mL / cm², at most about 9.6 with a cryoprotectant or as a lyophilizing agent includes mL / cm², at most about 9.5 mL/ cm², at most about 9.4 incubating the platelets for a time suitable for loading , as mL /cm² , at most about 9.3 mL/ cm², at most about 9.2 long as the time, taken in conjunction with the temperature , mL /cm² , at most about 9.1 mL /cm² , at most about 9.0 is sufficient for the cryoprotectant or lyophilizing agent to mL /cm² , at most about 8.9 mL / cm², at most about 8.8 come into contact with the platelets and , preferably , be mL /cm² , at most about 8.7 mL / cm², at most about 8.6 , incorporated , at least to some extent, into the platelets . In mL / cm2 at most about 8.5 mL / cm², at most about 8.4 some embodiments , incubation is carried out for about 1 mL / cm², at most about 8.3 mL / cm², at most about 8.2 minute to about 180 minutes or longer. mL /cm² , at most about 8.1 mL /cm² , at most about 8.0 [0270 ] The step of incubating the platelets to load them mL /cm² , at most about 7.9 mL / cm², at most about 7.8 with a cryoprotectant or lyophilizing agent includes incu mL/ cm², at most about 7.7 mL / cm², at most about 7.6 bating the platelets and the cryoprotectant at a temperature mL / cm², at most about 7.5 mL /cm² , at most about 7.4 that, when selected in conjunction with the amount of time mL /cm² , at most about 7.3 mL /cm² , at most about 7.2 allotted for loading , is suitable for loading. In general , the mL/ cm², at most about 7.1 mL / cm², at most about 6.9 composition is incubated at a temperature above freezing for mL/ cm², at most about 6.8 mL / cm², at most about 6.7 at least a sufficient time for the cryoprotectant or lyophiliz mL / cm², at most about 6.6 mL / cm², at most about 6.5 ing agent to come into contact with the platelets . In embodi mL / cm², at most about 6.4 mL /cm² , at most about 6.3 ments , incubation is conducted at 37 ° C. In certain embodi mL / cm², at most about 6.2 mL /cm² , at most about 6.1 ments , incubation is performed at 20 ° C. to 42 ° C. For mL/ cm² , at most about 6.0 mL / cm², at most about 5.9 example, in embodiments, incubation is performed at 35 ° C. mL /cm² , at most about 5.8 mL /cm² , at most about 5.7 to 40 ° C. ( e.g. , 37 ° C.) for 110 to 130 ( e.g. , 120 ) minutes . mL / cm², at most about 5.6 mL / cm², at most about 5.5 [0271 ] In various embodiments , the bag is a gas -perme mL / cm², at most about 5.4 mL/ cm², at most about 5.3 able bag configured to allow gases to pass through at least mL /cm² , at most about 5.2 mL /cm² , at most about 5.1 a portion or all portions of the bag during the processing . mL /cm² , at most about 5.0 mL /cm² , at most about 4.9 The gas- permeable bag can allow for the exchange of gas mL /cm² , at most about 4.8 mL /cm² , at most about 4.7 within the interior of the bag with atmospheric gas present mL / cm², at most about 4.6 mL / cm ?, at most about 4.5 in the surrounding environment. The gas -permeable bag can mL /cm² , at most about 4.4 mL/ cm², at most about 4.3 be permeable to gases, such as oxygen , nitrogen , water, air , mL /cm² , at most about 4.2 mL /cm² , at most about 4.1 hydrogen , and carbon dioxide , allowing gas exchange to mL /cm² , or at most about 4.0 mL /cm2 . In some embodi occur in the compositions provided herein . In some embodi ments , the SAN ratio of the container can range from about ments , the gas- permeable bag allows for the removal of 2.0 to about 10.0 mL/ cm² ( e.g., from about 2.1 mL /cm² to some of the carbon dioxide present within an interior of the about 9.9 mL / cm², from about 2.2 mL / cm² to about 9.8 bag by allowing the carbon dioxide to permeate through its mL /cm² , from about 2.3 mL /cm² to about 9.7 mL /cm² , from wall . In some embodiments , the release of carbon dioxide about 2.4 mL /cm² to about 9.6 mL / cm², from about 2.5 from the bag can be advantageous to maintaining a desired mL / cm² to about 9.5 mL /cm² , from about 2.6 mL/ cm² to pH level of the composition contained within the bag . about 9.4 mL / cm², from about 2.7 mL /cm² to about 9.3 [0272 ] In some embodiments , the container of the process mL / cm², from about 2.8 mL /cm² to about 9.2 mL /cm² , from herein is a gas- permeable container that is closed or sealed . about 2.9 mL / cm² to about 9.1 mL / cm², from about 3.0 In some embodiments , the container is a container that is mL /cm2 to about 9.0 mL/ cm², from about 3.1 mL /cm2 to closed or sealed and a portion of which is gas - permeable . In about 8.9 mL /cm² , from about 3.2 mL /cm² to about 8.8 some embodiments , the surface area of a gas- permeable mL / cm², from about 3.3 mL /cm2 to about 8.7 mL /cm² , from portion of a closed or sealed container ( e.g. , bag ) relative to about 3.4 mL /cm² to about 8.6 mL / cm², from about 3.5 the volume of the product being contained in the container mL / cm² to about 8.5 mL / cm², from about 3.6 mL / cm² to (hereinafter referred to as the “ SAN ratio ” ) can be adjusted about 8.4 mL / cm², from about 3.7 mL/ cm² to about 8.3 US 2020/0206143 A1 Jul . 2 , 2020 19 mL /cm² , from about 3.8 mL / cm² to about 8.2 mL / cm², from 1,1 -cyclopentanediacetic ( 3,3 tetramethylene - glutaric acid) ; about 3.9 mL / cm² to about 8.1 mL / cm², from about 4.0 piperazine -1,4 -bis- (2 - ethanesulfonic acid ) (PIPES ); N-( 2 mL / cm² to about 8.0 mL / cm², from about 4.1 mL / cm² to acetamido )-2 - amnoethanesulfonic acid ( ACES) ; 1,1- cyclo about 7.9 mL / cm², from about 4.2 mL / cm² to about 7.8 hexanediacetic ; 3,6 - endomethylene -1,2,3,6 - tetrahy mL / cm², from about 4.3 mL / cm² to about 7.7 mL / cm2, from drophthalic acid ( EMTA ; ENDCA ); imidazole ; about 4.4 mL / cm² to about 7.6 mL / cm², from about 4.5 2 - aminoethyl) trimethylammonium chloride mL / cm² to about 7.5 mL /cm² , from about 4.6 mL / cm² to ( CHOLAMINE) ; N , N - bis ( 2 - hydroxyethyl ) -2 - aminoethane about 7.4 mL / cm², from about 4.7 mL / cm² to about 7.3 sulfonic acid (BES ) ; 2 -methylpropane - 1,2,3 - triscarboxylic mL / cm², from about 4.8 mL /cm² to about 7.2 mL /cm² , from (beta -methyltricarballylic ) ; 2-( N -morpholino ) propane - sul about 4.9 mL /cm² to about 7.1 mL /cm² , from about 5.0 fonic acid (MOPS ); phosphoric ; and N - tris (hydroxymethyl ) mL / cm² to about 6.9 mL / cm², from about 5.1 mL /cm² to methyl- 2 - amminoethane sulfonic acid ( TES ). about 6.8 mL / cm², from about 5.2 mL / cm² to about 6.7 [0278 ] Flow cytometry is used to obtain a relative quan mL /cm² , from about 5.3 mL /cm² to about6.6 mL /cm2 , from tification of loading efficiency by measuring the mean about 5.4 mL/ cm² to about 6.5 mL/ cm², from about 5.5 fluorescence intensity of the drug in the drug -loaded plate mL/ cm² to about 6.4 mL/ cm², from about 5.6 mL / cm² to lets . Platelets are evaluated for functionality by ADP and / or about 6.3 mL / cm², from about 5.7 mL / cm² to about 6.2 TRAP stimulation post- loading . mL / cm² , or from about 5.8 mL/ cm² to about 6.1 mL / cm². [0279 ] In some embodiments the drug -loaded platelets are [0273 ] Gas- permeable closed containers ( e.g., bags ) or lyophilized . In some embodiments the drug - loaded platelets portions thereof can be made of one or more various are cryopreserved . gas -permeable materials . In some embodiments , the gas [0280 ] In some embodiments the drug - loaded platelets permeable bag can be made of one or more polymers retain the loaded drug upon rehydration and release the drug including fluoropolymers ( such as polytetrafluoroethylene upon stimulation by endogenous platelet activators . (PTFE ) and perfluoroalkoxy (PFA ) polymers ), polyolefins [0281 ] In some embodiments the dried platelets (such as ( such as low -density polyethylene ( LDPE ) , high -density freeze -dried platelets ) retain the loaded drug upon rehydra polyethylene (HDPE ) ) , fluorinated ethylene propylene tion and release the drug upon stimulation by endogenous ( FEP ), polystyrene, polyvinylchloride (PVC ) , silicone, and plateletactivators . In some embodiments at least about 10 % , any combinations thereof. such as at least about 20 % , such as at least about 30 % of the [0274 ] In some embodiments the lyophilizing agent as drug is retained . In some embodiments from about 10 % to disclosed herein may be a high molecular weight polymer . about 20 % , such as from about 20 % to about 30 % of the By “ high molecular weight” it is meant a polymer having an drug is retained . average molecular weight of about or above 70 kDa and up [ 0282 ] An example of a drug that may be loaded in a to 1,000,000 kDa. Non -limiting examples are polymers of platelet is doxorubicin . Another example is of a drug that sucrose and epichlorohydrin (poly sucrose ). Although any may be loaded in a platelet is olaparib . Another example of amount of high molecular weight polymer can be used , it is a drug that may loaded in a platelet is paclitaxel . preferred that an amount be used that achieves a final [ 0283 ] Various agents and / or procedures may be used to concentration of about 3 % to 10 % (w /v ), such as 3 % to 7 % , load the platelets with a drug. In some embodiments , the for example 6 % . Other non -limiting examples of lyopro platelets are loaded with a liposomal formulation of the tectants are serum albumin , dextran , polyvinyl pyrrolidone drug . In some embodiments , the drug is not comprised in a (PVP ) , starch , and hydroxyethyl starch (HES ) . liposomal formulation . In some embodiments , the platelets [0275 ] In some embodiments , the loading buffer com are loaded with a drug previously incubated with a cell prises an organic solvent, such as an alcohol ( e.g. , ethanol ) . penetrating peptide. In some embodiments , the platelets are In such a loading buffer , the amount of solvent can range loaded with a drug previously incubated with a cationic lipid from 0.1 % to 5.0 % ( v / v ) . such as lipofectamine . In some embodiments , the platelets [0276 ] In some embodiments the drug - loaded platelets are loaded with the drug in the presence of a detergent. For prepared as disclosed herein have a storage stability that is example, the detergent may be saponin . at least about equal to that of the platelets prior to the loading [0284 ] In some embodiments , the platelets are loaded by of the drug . a process comprising endocytosis . [0277 ] The loading buffer may be any buffer that is [0285 ] In some embodiments , the platelets are loaded by non -toxic to the platelets and provides adequate buffering a process comprising electroporation . capacity to the solution at the temperatures at which the [0286 ] In some embodiments , the platelets are loaded by solution will be exposed during the process provided herein . a process comprising transduction . Thus, the buffer may comprise any of the known biologi [0287 ] In some embodiments , the platelets are loaded by cally compatible buffers available commercially , such as a process comprising sonoporation . phosphate buffers, such as phosphate buffered saline (PBS ) , [0288 ] In some embodiments , the platelets are loaded by bicarbonate /carbonic acid , such as sodium - bicarbonate buf a process comprising osmotic hypertonic /hypotonic loading / fer , N - 2 -hydroxyethylpiperazine - N '- 2 - ethanesulfonic acid hypotonic shock . Hypotonic shock uses a solution with (HEPES ) , and tris - based buffers , such as tris -buffered saline lower osmotic pressure to induce cell swelling leading to ( TB S ). Likewise, it may comprise one or more of the membrane permeability . Hypertonic shock may increase following buffers : propane- 1,2,3 -tricarboxylic ( tricarbally platelet loading of cryoprotectants or lyoprotectants ( e.g., lic ) ; benzenepentacarboxylic ; maleic ; 2,2 -dimethyl succinic ; trehalose) (Zhou X., et . al. , Loading Trehalose into Red EDTA ; 3,3 -dimethylglutaric ; bis (2 -hydroxyethyl ) imino - tris Blood Cells by Improved Hypotonic Method , Cell Preser (hydroxymethyl ) -methane (BIS - TRIS ); benzenehexacar vation Technology , 6 ( 2) , https://doi.org/10.1089/cpt.2008 . boxylic (mellitic ) ; N-( 2 -acetamido ) imino -diacetic acid 0001 ( 2008 ) , which is herein incorporated by reference ) . (ADA ) ; butane - 1,2,3,4 - tetracarboxylic ; pyrophosphoric ; Additionally and alternatively , hypotonic shock may allow US 2020/0206143 A1 Jul. 2. 2020 20 the uptake and internalization of large and /or charged mol cell- free Pep - 1 : protein complex . Loaded platelets may then ecules through passive means, such as, endocytosis , micro be lyophilized to make Thrombosomes. Platelets that have pinocytosis , and /or diffusion . accumulated Pep - 1 can be detected via flow cytometry or [0289 ] In some embodiments , the solutes in the hypertonic fluorescence microscopy if a fluorescent tag is attached to solution can be, in a non - limiting way, salts , low -molecular the C -terminus cysteamine of Pep - 1. If the cargo protein is weight sugars ( e.g. , monosaccharides, disaccharides ) , or low fluorescently labeled , then platelets containing this cargo molecular weight inert hydrophilic polymers. may also be detected using flow cytometry or fluorescence [ 0290 ] In some embodiments , the platelets are loaded by microscopy . a process comprising the use of Transfection Reagents ( also [0298 ] The HIV Tat protein is another example of a cell described in WO2014118817A2 , incorporated by reference penetrating peptide. The Tat protein includes between 86 herein in its entirety ). and 101 amino acids depending on the subtype . Tat is a [0291 ] Exemplary protocols that employ the foregoing regulatory protein that enhances the viral transcription effi agents or procedures are described below : ciency . Tat also contains a protein transduction domain [ 0292 ] A liposome is a vesicle made of phospholipid which functions as a cell- penetrating domain allowing Tat to bilayer . This vesicle can be designed to encapsulate drug of cross cellular membranes . interest , which is delivered inside a cell following the fusion [0299 ] Lipofectamine is a cationic lipid ; the Lipo of vesicle and cell membrane . fectamine positively charged head group interacts with the [0293 ] Liposome encapsulated Doxorubicin ( chemo negatively charged phosphate backbone of nucleic acids to therapy drug ) is prepared through rehydration of lyophilized facilitate transfection . Cellular internalization of the nucleic lipids (Sigma - Aldrich , L4395-1VL ) with drug in PBS fol acid is achieved by incubating cells with the complexed lowed by 30 seconds of agitation via vortex , then 30 minutes Lipofectamine and nucleic acid . of incubation at 37 ° C. The liposommes are then incubated [ 0300) Prepare the Lipofectamine and nucleic acid com with platelets at 37 ° C. for 30 minutes. Cells are washed plex in aqueous buffer at room temperature . Incubate the once via centrifugation to remove incorporated liposome complexed Lipofectamine and nucleic acid with platelets for encapsulated doxorubicin or free doxorubicin . Drug loaded 2-3 hours . Transfected platelets may be lyophilized to create platelets can be lyophilized in appropriate buffer to create Thrombosomes. Fluorescently labeled nucleic acid can be Thrombosomes. Flow cytometry and fluorescence micros detected via flow cytometry and visualized using fluores copy may be performed to assess drug loading and intrac cence microscopy . This method of loading is applicable to ellular localization . A fluorescence microplate reader can be both RNA and DNA . used to obtain quantification of drug load . Light transmis [0301 ] An electroporation machine generates electrical sion aggregometry will be used to evaluate platelet function pulses which facilitate formation of transient openings in post drug load . plasma membranes . The increased plasma membrane per [0294 ] Endocytosis is a process through which a cell takes meability allows entry of large and / or charged cargo that in material from its surroundings. The cell invaginates its would otherwise not enter the cell due to membrane barrier . plasma membrane to wrap around fluid or particles in its [0302 ] Perform electroporation of platelets in the presence immediate environment. The internalized vesicle buds off of desired cargo . Cargos of interest can be detected by flow from the plasma membrane and remains inside the cell . cytometry and fluorescence microscopy if they are fluores [0295 ] Co -incubation of platelets with drug of interest cently tagged occurs at 37° C. for 1-4 hours during which drug is loaded [ 0303 ] The influx cell loading strategy harnesses osmosis into platelets via endocytosis . Loaded platelets may then be to load cells with water soluble, polar compounds. Cells are lyophilized to make Thrombosomes . Loaded drug is initially placed in a hypertonic solution containing drug of detected via flow cytometry or fluorescence microscopy , interest . In this hypertonic solution , water will move out of provided drug is fluorescently tagged or is itself fluorescent. the cell into solution while drug will move into the cell via Loaded drug can be detected by HPLC or a microplate pinocytosis . Following that , cells are placed in a hypotonic reader ( e.g. , a Tecan plate reader ). Endocytic inhibitors such solution in which water will enter the cell , lysing the as amiloride ( 1 mM ), phenylarsine oxide ( 10 uM ), cytocha pinocytic vesicles and thereby releasing drug into the cyto lasin D ( 4 uM ), or dynasore ( 25 uM ) can be used to confirm sol. that platelet loading is achieved by endocytosis . [0304 ] Incubate platelets in hypertonic loading medium [ 0296 ] Pep - 1 is a 21 amino acid cell penetrating peptide containing drug compound at 37 ° C. for at least 1 hour. with a C - terminal cysteamine group that shuttles cargo such Isolate loaded platelets from solution via centrifugation , as proteins or peptides into target cells . Pep - 1 consists of a resuspend platelets in hypotonic lysis medium , and incubate hydrophobic domain linked to a hydrophilic domain . The at 37 ° C. Pinocytic vesicles will burst and release drug into hydrophobic , tryptophan - rich domain can associate with a the cytosol. Fluorescently labeled drug can be visualized target cell membrane and the hydrophobic domains of the using fluorescence microscopy to confirm internalization . cargo protein (See e.g., Heitz , F., et . al ., Twenty years of Flow cytometry may be performed to quantify drug load per cell- penetrating peptides: from molecular mechanisms to cell for fluorescent drug . therapeutics , British Journal of Pharmacology, 157, 195 [ 0305 ] Viral vectors are commonly used for transduction 206 , (2009 ) , which is incorporated herein by reference in its of cells . The host cell is driven by the viral vector to express entirety ). the protein of interest at high load . [0297 ] The Pep -1 and the cargo protein are complexed by [0306 ] Use lentiviral vector to transfect 293T cells to co - incubation at 37° C. for 30 minutes . The Pep -1 : protein generate pseudovirus, which is collected from the superna complex is incubated with platelets at 37 ° C. for at minimum tant of this cell culture . The pseudovirus is then used to 1 hour to allow Pep - 1 mediated loading of protein cargo into transduce megakaryocytes. Inside the transduced mega the platelet. Platelets are washed by centrifugation to remove karyocyte , viral core plasmid containing cytomegalovirus US 2020/0206143 A1 Jul . 2 , 2020 21 promotor drives overexpression of the protein of interest, the therapeutic indications for cargo to be loaded into which gets packaged into platelets that bud off from trans platelets include, for example , targeted depletion of cancer duced megakaryocytes . cells with chemotherapy drugs and therapeutic or prophy [0307 ] Human platelets express FcyRIIA receptor which lactic treatment of bacterial infection at site of injury with antibiotics . binds to the Fc region of IgG and facilitates internalization [0312 ] In some embodiments , provided herein is a method of IgG immune complexes. This method of loading platelets of treating a disease as disclosed herein , comprising admin provides a route for delivery of therapeutic antibodies. istering drug - loaded platelets , drug - loaded platelet deriva [0308 ] Incubate fluorescently labeled IgG at 62 ° C. for 20 tives, or drug -loaded thrombosomes as disclosed herein . In minutes to prepare IgG immune complexes . Incubate IgG some embodiments , provided herein is a method of treating immune complexes with platelets for 1 hour at 4 ° C. to allow a disease as disclosed herein , comprising administering cold cells to bind immune complexes. Next, incubate immune stored, room temperature stored , cryopreserved thawed , complex -bound platelets at 37 ° C. to allow internalization of rehydrated , and /or lyophilized platelets, platelet derivatives, immune complexes. Flow cytometry can detect internalized or thrombosomes as disclosed herein . fluorophore labeled IgG immune complexes. An anti - IgG [ 0313 ] Examples of diseases ( therapeutic indications ) that PE antibody specific for immune complexes can be used to may be treated with the drug - loaded platelets are as follows: identify surface bound , but not internalized , IgG - FITC immune complex . [ 0309 ] Examples of drugs and of loading agents are as Therapeutic indications follows: Acute lymphoblastic leukemia ( ALL ) Acute myeloid leukemia ( AML ) Breast cancer (can also be used as an Osmotic adjuvant therapy for metastasized Cell hypertonic / breast cancer post- surgery ) penetrating hypotonic Gastric cancer Endocytosis peptide loading Hodgkin lymphoma Neuroblastoma Dextran 10K Dextran 10K Dextran 10K Non - Hodgkin lymphoma Dextran 500K Dextran 3K Dextran 3K Ovarian cancer FITC - Albumin Dextran 500K Dextran 500K Small cell lung cancer FITC - Bovine IGG FITC - albumin FITC - albumin Soft tissue and bone sarcomas FITC - F ( ab ) 2 FITC - Bovine IGG FMLP Thyroid cancer Histone H1 FITC - F ( ab ) 2 Histone H1 Transitional cell bladder cancer Lucifer yellow FMLP Lucifer Yellow Wilms tumor slow uptake Cancer PE Histone H1 PE (PHYCOERYTRIN ) Rabbit IGG Lucifer yellow Rabbit IGG [0314 ] Examples of cargo and therapeutic indications for Soybean Trypsin PE Soybean Trypsin cargo (s ) to be loaded into platelets are as follows: Inhibitor Inhibitor Doxorubicin Rabbit IGG Doxorubicin Olaparib Soybean Trypsin Inhibitor Cargo Therapeutic indications Paclitaxel Chemotherapy Acute lymphoblastic leukemia (ALL ) drug Acute myeloid leukemia (AML ) ( e.g., DOX , Breast cancer ( can also be used as an adjuvant [0310 ] In some embodiments , the loading step comprises Olaparib , therapy for metastasized breast cancer post- surgery ) the use of dextran as a lyophilizing agent . In some embodi Paclitaxel) Cancer Gastric cancer ments the drug is an antibody. In some embodiments when Hodgkin lymphoma the drug is an antibody, the drug is labeled with FITC Neuroblastoma ( fluorescein isothiocyanate or 3 ', 6 '- dihydroxy - 6 - isothiocya Non -Hodgkin lymphoma natospiro [ 2 - benzofuran -3,9 '- xanthene ]-1 -one ). Ovarian cancer Small cell lung cancer [0311 ] In some embodiments , drug - loaded platelets , drug Soft tissue and bone sarcomas loaded platelet derivatives, or drug- loaded thrombosomes Thyroid cancer may shield the drug from exposure in circulation , thereby Transitional cell bladder cancer reducing or eliminating systemic toxicity ( e.g. cardiotoxic Wilms tumor ity ) associated with the drug. In some embodiments , drug loaded platelets , drug -loaded platelet derivatives , or drug [0315 ] In some embodiments , a drug may be fluorescent loaded thrombosomes may also protect the drug from or labeled with a fluorescent moiety . For such a fluorescent metabolic degradation or inactivation . In some embodi or labeled drug , a correlation may be established between ments, drug delivery with drug - loaded platelets , drug - loaded the fluorescence intensity and its concentration , and such a platelet derivatives, or drug - loaded thrombosomes may correlation may then be used to determine the concentration therefore be advantageous in treatment of diseases such as of the drug over a range of values . For example , FIG . 7 cancer , since drug - loaded platelets , drug - loaded platelet illustrates the correlation between fluorescence and concen derivatives, or drug - loaded thrombosomes facilitate target tration for doxorubicin . As FIG . 7 shows, the value of the ing of cancer cells while mitigating systemic side effects . In concentration for doxorubicin in uM is determined accord some embodiments , drug - loaded platelets , drug - loaded ing to the equation platelet derivatives, or drug - loaded thrombosomes may be used in any therapeutic setting in which expedited healing X = ( Y + 344.92 )/996.6 process is required or advantageous. In some embodiments , where Y is the fluorescence and X is the concentration . US 2020/0206143 A1 Jul . 2 , 2020 22

[0316 ] An analogous correlation can be derived for the TABLE 3 - continued value of the amount of doxorubicin in mg: Buffer B mg of doxorubicin = concentration (umol / L )x543.42 g /molx50 ul/ well / 10 % Concentration (MM unless otherwise or Component specified ) mg of doxorubicin = (# umol DOX / L ) (mol / 10 umol) Dextrose 5 ( 543.52 g /mol ) ( 103 mg/ g ) ( L / 103 ml) ( 1 ml/ 103 pH 7.4 ml) (50 ul/ well ) [ 0317 ] An analogous correlation can be derived for the [ 0322 ] Table 3. Buffer B is used when incubating plate value of the amount of doxorubicin in mg/ cell: lets with fluorophore conjugated antibodies for flow mg/ cell =mg ( Intracellular + Membrane -bound doxoru cytometry . This incubation is done at room temperature bicin ) / total # of cells in a well. in the dark . [ 0318 ] Thus , the concentration of doxorubicin may be [ 0323 ] Albumin is an optional component of Buffer B quantified from its excitation /emission spectra . ( 0319 ] Examples of loading buffer that may be used are TABLE 4 shown in Tables 1-4 : Concentration of TABLE 1 HEPES and of Salts in Buffer B Concentration (mM Loading Buffer unless otherwise Concentration (mM Component specified ) unless otherwise HEPES 25 Component specified ) NaCl 119 5 75.0 ??? NaCl 2 KCI 4.8 CaCl2 2 HEPES 9.5 MgCl2 NaHCO3 12.0 Glucose 6 g /l Dextrose 3 Trehalose 100 Ethanol 1 % ( v / v ) [ 0324 ] In Table 4 the pH adjusted to 7.4 with NaOH [0325 ] Albumin is an optional component of Buffer B [0320 ] Table 1. Loading Buffer is used to load platelets [0326 ] In some embodiments, drug - loaded platelets are via endocytosis at 37 ° C. with gentle agitation as prepared by incubating the platelets with the drug in a sample is placed on a rocker. Adjust pH to 6.6-6.8 loading buffer having the components shown in the table below . TABLE 2 [ 0327 ] In some embodiments , the loading buffer has the Buffer A components as listed above in Table 1 . [0328 ] In some embodiments , incubation is performed at Concentration (mM unless specified 37 ° C. using a platelet to drug volume ratio of 1 : 2 , using Component otherwise ) different incubation periods. CaCl2 1.8 MgCl2 1.1 Example 1. Doxorubicin - Loaded Platelets KC1 2.7 NaCl 137 [0329 ] Doxorubicin - loaded platelets were prepared by NaH2PO4 0.4 incubating the platelets with doxorubicin in a loading buffer HEPES 10 having the components shown in Table 5. Protocol 1 (de D - glucose 5.6 pH 6.5 scribed below ) was used . [0330 ] The platelet concentration in the loading buffer was [0321 ] Table 2. Buffer A is used for loading platelets 200,000 platelets / ul. The loading buffer had the following with liposome encapsulated drug . Incubation done at components : 37 ° C. with gentle agitation as sample is placed on a rocker . TABLE 5 Concentration TABLE 3 Component (MM unless specified otherwise) Buffer B NaCl 75.0 KCI 4.8 Concentration (mM HEPES 9.5 unless otherwise NaHCO3 12.0 Component specified ) Dextrose 3 Trehalose 0.1M Buffer and Salts Table 4 (below ) Ethanol 1 % (v /v ) BSA 0.35 % US 2020/0206143 A1 Jul . 2 , 2020 23

[0331 ] Incubation was performed at 37 ° C. using a platelet centration of 13.5 uM . Doxorubicin was efficiently loaded to drug volume ratio of 1 : 2 , using different incubation after 0.5 hours of incubation with the doxosomes . periods. [0339 ] As can be seen from the Figures, loading of doxo [0332 ] The drug - loading method was evaluated by flow rubicin occurred efficiently both via doxosomes (FIG . 5 ) and cytometry to obtain a relative quantification of loading using endocytotic loading (FIGS . 4A -4C ) . efficiency as mean fluorescence intensity of Doxorubicin in [0340 ] Exemplary protocols for the loading of platelets drug - loaded platelets . Platelets were evaluated for function with doxorubicin are shown below : by ADP and /or TRAP stimulation post- loading. The result ing amounts of doxorubicin load as a function of incubation [0341 ] Protocol 1. Loading Platelets with DOX Via Endo time are provided in FIG . 1. Platelets were loaded with cytosis fluorescent DOX ( excitation 470 nm , emission 560 nm ) and [0342 ] The starting apheresis platelet material was pooled evaluated by flow cytometry for fluorescence uptake . and characterized . The platelet pool was acidified to pH CD42b + platelets load increasingly more DOX over time. % 6.6-6.8 using Acid Citrate Dextrose solution . Platelets were of platelets loaded with DOX is > 90 % , as shown in FIG . 4 . isolated by centrifugation at 1500 g for 20 minutes , with DOX = doxorubicin . N = 1 . slow acceleration and braking . The supernatant plasma was [0333 ] In FIG . 2 , DOX loaded platelets were incubated in aspirated and disposed of. loading buffer with ADP and / or TRAP for 10 minutes at [0343 ] The platelets were suspended in the loading buffer room temperature to stimulate drug release . Following this of Table 1 at a concentration of 200,000 platelets / ul. While incubation , flow cytometry was performed to assess the platelets were being centrifuged , a doxorubicin (“ DOX ” ) decrease in drug load . DOX + populations were gated on solution having a concentration of 0.3 mM in the loading CD42b + platelets . TRAP partially induced DOX release buffer was prepared as follows: a solution of DOX in water from drug -loaded platelets while ADP did not. (50 mg/ ml ) was pre -warmed at 37 ° C. for 20 minutes; DOX [ 0334 ] There is no synergistic effect of TRAP and ADP on was then incubated in the loading buffer at a concentration DOX release from loaded platelets . ADP = adenosine diphos of 0.3 mM at 37 ° C. for 20 minutes , periodically subjecting phate ; TRAP = thrombin receptor activating peptide ; it to a vortex . DOX = doxorubicin . N = 1 . [0344 ] The platelets and DOX (1 ml platelets at 200,000 [0335 ] In FIG . 3, platelets (200,000 platelets /ul ) were platelets /ul + 2 ml DOX at 0.3 mM ) were then mixed and the loaded with liposome encapsulated DOX (2 mg/ ml ) . Lipo mixture was incubated at 37 ° C. for 2 hours . The resulting some encapsulated DOX was prepared by rehydrating DOX - loaded platelets were lyophilized . lyophilized lipid mixture ( from Liposome Kit, Sigma- Al [0345 ] Optionally , the lyophilized DOX -loaded platelets drich , L4395 ) with 2 mg/ml DOX in PBS , followed by could be suspended in water at a concentration suitable for incubation at 37 ° C. for 30 minutes . Platelets and liposome the uses disclosed herein . encapsulated DOX were incubated at 37 ° C. for 30 minutes [0346 ] Protocol 2. Loading Platelets with Liposome in Tyrode's HEPES buffer ( Table 2 ). Flow cytometry was Encapsulated DOX performed to obtain qualitative quantification of amount of [0347 ] The starting apheresis platelet material was pooled DOX loaded per platelet . % of CD42b + platelets loaded with and characterized . The platelet pool was acidified to pH liposome encapsulated DOX was > 90 % as shown in FIG . 5 . 6.6-6.8 using Acid Citrate Dextrose solution . Platelets were N = 1 . isolated by centrifugation at 1500 g for 20 minutes , with slow acceleration and braking . The supernatant plasma was Example 2. Time Course of Endocytic Loading Vs. aspirated and disposed of. Liposome- Mediated Loading of Doxorubicin into [0348 ] The platelets were suspended in Buffer A at a Platelets concentration of 200,000 platelets /ul . The components of [ 0336 ] The efficiency of Doxorubicin loading in to plate Buffer A are shown above Table 2 . lets via standard endocytosis or using liposome- encapsu lated Doxorubicin (“ Doxosomes” ) was tested . TABLE 6 [0337 ] FIGS. 4A - 4C provide data relating to endocytic loading of Doxorubicin into platelets . Platelets were incu Tyrode's HEPES Buffer bated with 0.1 mM Doxorubicin or 0.3 mM Doxorubicin for two hours . Doxorubicin was allowed to enter the platelets Component Concentration (MM ) via endocytosis . The top graph shows amalgamated data that CaCl2 1.8 MgCl2 1.1 includes the bottom left graph (Doxorubicin at 0.1 mM ) and KCI 2.7 the bottom right graph (Doxorubicin at 0.3 mm ) . Doxoru NaCl 137 bicin was efficiently loaded after two hours of incubation at NaH2PO4 0.4 concentrations of both 0.1 mM and 0.3 mm . For each graph , HEPES 10 the left- most curve represents cells only . D - glucose 5.6 [0338 ] FIG . 5 provides data relating to liposome- mediated pH 6.5 loading of Doxorubicin into platelets . Platelets were incu bated with 13.5 uM of Doxorubicin - containing doxosomes [0349 ] While the platelets were being centrifuged , lipo for 0.5 hours . Briefly , a total of 90 umol of lipids (63 umol some- encapsulated doxorubicin (“ DOX ” ) was prepared as L - A -Phosphatidylcholine , 18 umol Stearylamine, and 9 follows: lyophilized phospholipids (Sigma - Aldrich , SKU # ?mol Cholesterol) were resuspended in 1 mL of Doxorubicin DOX - 1000 ) were rehydrated with a 2 mg/ ml solution of in PBS to generate the doxosomes . 150 uL of doxosome DOX in PBS ; the rehydrated mixture was then subjected it containing 12 mg/ mL of liposome- encapsulated Doxorubi to a vortex for 30 seconds and incubated at 37 ° C. for 30 cin was incubated with platelets to a final doxosome con minutes. US 2020/0206143 A1 Jul . 2 , 2020 24

[0350 ] The platelets in Tyrode's HEPES buffer and the in a rocker at low frequency, in the presence of a buffer liposomal DOX were then mixed and the mixture was containing HMT and 1 uM PGE1. The correlation between incubated at 37 ° C. for 30 minutes . fluorescence and concentration is shown in FIG . 7 . [0351 ] The resulting DOX -loaded platelets were washed in 1 mL Tyrode's HEPES buffer to remove unincorporated Calculations of Amounts — Example 3A - 1 liposome by centrifugation at 1500 g for 20 minutes. [0352 ] Optionally , the lyophilized DOX - loaded platelets [0360 ] 1 ) Concentration : could be suspended in water at a concentration suitable for [0361 ] In a well containing 50 ul and having a con the uses disclosed herein . centration of 250K cells per the intracellular doxoru [0353 ] Protocol 3. Loading Platelets with Liposome bicin fluorescence was 10811 and the membrane -bound Encapsulated DOX doxorubicin fluorescence was 1263. Calculating the [ 0354 ] This protocol was similar to Protocol 2 herein , concentration from the formula except that the buffer was as follows instead of the buffer of X = ( Y + 344.92 )/996.6 , Table 6 above : [0362 ] where Y is the fluorescence and X is the con TABLE 7 centration , provides an intracellular concentration 11 UM and a membrane -bound concentration of 2 uM . Tyrode's HEPES Buffer (plus PGE1 ) [0363 ] 2 ) Quantity in mg : Component Concentration (MM ) [0364 ] The amount in mg is calculated from the for mula : CaCl2 1.8 MgCl2 1.1 mg of doxorubicin = concentration (umol / L )x543.42x ??? 2.7 50/109 NaCl 137 NaH2PO4 0.4 [ 0365 ] 3 ) Quantity in mg/ Platelet: HEPES 10 D - glucose 5.6 [0366 ] The amount in mg/ platelet is calculated from the pH 6.5 formula : Prostagalandin 1 ug /ml ( Intracellular DOX amount (per well) +membrane E1 (PGE1 ) bound DOX amount (per well )) / total # of plate lets in a well [ 0355 ) Protocol 4. Loading Platelets with Liposome [0367 ] For example , for an intracellular amount of Encapsulated DOX 3.04x10-4 mg and a membrane -bound amount of 4.38x [0356 ] This protocol was similar to protocol 2 herein , 1095 except that the buffer of Table 1 was used ( shown again mg , if there are 250,000 cells /ul and 50,1.1 /well , below ) instead of the buffer of Table 6 above : then the total amount will be 2.78x10-11 mg /platelet . Experimental Results . Effect of Buffer on Liposome -Medi [0368 ] The calculations have a percentage error of ated Loading of Doxorubicin into Platelets 7.16 % , calculated as follows: [0357 ] Platelets were incubated with doxosomes contain [0369 ] Intracellular concentration of doxorubicin : 11 ing fluorescent doxorubicin as described above in Example UM 2 in the presence of trehalose - containing loading buffer ( as [ 0370 ) membrane -bound concentration of doxorubicin : shown in Table 1 ) or HMT (as shown in Table 7 ) As can be 2 uM . seen in FIG . 6 , trehalose- containing loading buffer increased [0371 ] Soluble concentration of doxorubicin : 298 UM . the total amount of Doxorubicin that was loaded as com [0372 ] Total concentration of doxorubicin : 290 uM . pared to HMT. [0358 ] FIGS. 6A and 6B provide a comparison of doxo % error = [ (298 + 11 + 2 ) -290 ) /290 ]x100 % = 7.16 % some loading efficiency between conventional HMT buffer (Protocol 3 , shown in continuous line ) and trehalose - con Calculations of Amounts Example 3A - 2 taining loading buffer (Protocol 4 , shown in individual points ) described herein . The x axis represents the amount of [0373 ] 1) Concentration : doxosome added to the platelets . The y axis represents [0374 ] In a well containing 50 ul and having a con mean - fluorescence intensity . FIG . 6A provides the doxo centration of 125K cells per the intracellular doxoru some loading efficiency of loading platelets with CD42b bicin fluorescence was 7496 and the membrane -bound antibodies to define the platelets while FIG . 6B provides the doxorubicin fluorescence was 438. Calculating the con doxosome loading efficiency of platelets without CD42b . A centration from the formula higher drug load is obtained with the trehalose- containing loading buffer of Protocol 4 as compared to the conventional X = ( Y + 344.92 ) /996.6 , HMT loading buffer at all points after about 20 minutes . [0375 ] where Y is the fluorescence and X is the con centration , provides an intracellular concentration 8 uM Example 3 — Determination of Amount of and a membrane -bound concentration of 1 uM . Doxorubicin ( “ DOX ” ) in a Platelet [0376 ] 2 ) Quantity in mg: [0377 ] The amount in mg in a 50 uL sample is calcu Protocol A : lated from the formula : [0359 ] Fresh donor platelets were provided . Incubation mg of doxorubicin = concentration ( umol/ L )x543.42x with the drug took place by endocytosis at 37 C for 3 hours 50 / 10 ° US 2020/0206143 A1 Jul . 2 , 2020 25

[ 0378 ] 3 ) Quantity in mg/Platelet : isolated from buffer via centrifugation (Beckman Coulter [0379 ] The amount in mg/ platelet is calculated from the Microfuge 18 Centrifuge , 845xg, 10 minutes , room tem formula : perature ), then washed twice with LB or HMT using the ( Intracellular DOX amount+ membrane -bound DOX same centrifugation setting as stated . After that , the platelets amount )/ total # of platelets in a well were sonicated 3 times to release intracellular DOX at 26 kHz for 30 seconds with 2-5 minutes interval of rest at room [ 0380 ] For example , for an intracellular amount of temperature. These samples are then centrifuged (Eppendorf 2.14x10-4 mg and a membrane- bound amount of 2.13x Centrifuge 5424 , 18,000 G , 20 minutes , room temperature ) 10-5 , if there are 125,000 cells/ ul and 50 ul/ well , then to separate intracellular DOX ( supernatant) from membrane the total amount will be 1.88x1011 mg/ platelet . bound DOX (pellet ). The pellet was resuspended in 1 ml of [ 0381 ] The calculations have a percentage error of buffer (HMT or LB ) then sonicated 3 times at previously 1.94 % , calculated as follows: stated setting to generate homogenized sample of membrane [0382 ] Intracellular concentration of doxorubicin : 8 UM bound DOX in buffer . Quantification of DOX is achieved [0383 ] membrane -bound concentration of doxorubicin : with 500 nm excitation and 600 nm emission using the 1 uM . TECAN Infinite M200 PRO . A 96 -welled polystyrene, half [0384 ] Soluble concentration of doxorubicin : 295 uM . area , non - treated , black with clear flat bottom plate ( Corn [0385 ] Total concentration of doxorubicin : 310 UM . ing ) is used in which 504 , of sample is plated per well in % error = [ (295 + 8 + 1 ) -310 ) / 310 ]x100 % = 1.94 % triplicates . DOX load per platelet (mg / cell ) is calculated based on standard curve (linear regression , R2 = 0.9873 for Protocol B : cell lysate in HMT, R2 = 0.9902) generated from serial dilu tion of cell lysate . [0386 ] Four apheresis units were pooled. Incubation with [0394 ] As shown in FIG . 12 , the loading buffer allowed the drug took place at 37 C for 4 hours in a rocker at low higher drug load per cell than compared to HMT buffer . frequency, in the presence of a buffer containing HMT and 1 uM PGE1 . The correlation between fluorescence and Example 5 — Induced Drug Release from concentration is shown in FIG . 13 . Doxorubicin - Loaded Platelets [0395 ] Pooled apheresis platelets from between 8 and 11 Calculations of Amounts Example 3B - 1 apheresis units (defined as 200-400 mL of plasma from a [0387 ] 1 ) Concentration : single donor ) were diluted to 250,000 cells /?L . A test group [0388 ] Calculations were performed in a manner analo included 0.2 mM doxorubicin (DOX ) and a control group gous to Example 3A - 1 and 3A - 2 above , the correlation contained no agonist . Both the test and control groups were between fluorescence and concentration was based on incubated in a HMT buffer of pH 6.8 containing 1 UM a standard curve . Prostaglandin E1 ( PGE1) for 3 hours at 37 ° C. [0389 ] The resulting quantity of DOX in mg/ platelet for [0396 ] Any excess drug not loaded into the platelet was various values of cells /ul is as follows : removed by subjecting the test group to centrifugation at 800xg for 10 minutes at room temperature and by discarding the supernatant. The drug - loaded cells of the test group were Cells /ul DOX (mg /cell ) resuspended to the same concentration in HMT buffer at pH 108K 4.4 x 10-6 7.4 then stimulated with agonists including collagen , ADP, 235K 3.2 x 10-6 thrombin , arachidonic acid (AA ) and thrombin rece tor 454K 2.2 x 10-6 915K 1.5 x 10-6 activating peptide ( TRAP - 6 ) for 15 minutes at room tem 1448K 1.0 x 10-7 perature . The supernatant was harvested by centrifugation as 1745K 9.3 x 10-7 described above . [ 0397 ] The control group for intracellular drug was pre pared by three rounds of vibration sonication for 30 seconds [0390 ] FIGS. 8 and 9 are based on the same data showing at 20 kHz prior to centrifugation . that the total amount ofDOX loaded into platelets increases [0398 ] Fifty ul of supernatant of the test and control as a function of increasing platelet count, but the amount of groups was applied to fluorescent compatible clear flat DOX loaded into an individual platelet decreases. bottom 1/2 area of 96 well micro - titer plates and analyzed on [0391 ] FIG . 8 shows the total concentration of (a ) intrac a Tecan Sapphire plate reader with excitation at 500 nm and ellular doxorubicin , and (b ) membrane -bound doxorubicin , emission at 600 nm and the released Doxorubicin quantified with increasing concentration of platelets /uL . relative to a standard curve obtained under the same condi [0392 ] FIG . 9 shows how for a given concentration of tions . doxorubicin (DOX ) (0.2 mm ) , the amount of DOX /platelet [0399 ] The amount of released and intracellular DOX in decreases as the number of platelets increases . DOX - loaded platelets when stimulated with varying amounts of collagen are shown in FIG . 10. The effect of Example 4. Comparison of Amount of Drug varying amounts of collagen on inducing drug release on Loaded with Loading Buffer and with HMT Buffer DOX - loaded platelet , as measured by light excitation and [0393 ] Platelets pooled from eight apheresis units , at a emission , is shown in FIG . 10 in comparison with a control concentration of (250,000 cells /uL , were incubated in either group containing no collagen . the Loading Buffer of Table 1 (LB in FIG . 12 ) or HMT [0400 ] The amount of released and intracellular DOX in containing PGE1 ( 1 uM ) ( Table 7) in the presence of DOX DOX - loaded platelets when stimulated with TRAP - 6 , ADP , ( 0.6 mM or 0.36 mg/ ml) compared to a controlwith no DOX AA , and thrombin are shown in FIG . 11. The effect of for 3 hours at 37 ° C. Following incubation , platelets were different agonists on inducing drug release on DOX -loaded US 2020/0206143 A1 Jul. 2. 2020 26

platelet, as measured by light excitation and emission , is [ 0408 ] A series of quality control experiments were per shown in FIG . 11 in comparison with a control group formed with DOX loaded thrombosomes. The batch is a containing no agonist. large batch measured in apheresis units . The batch contains Sublot A with 98 vials of unloaded thrombosomes and Example 6 - Loading Buffer Allows Higher Drug Sublot B with 98 vials of DOX - loaded thrombosomes . Load Per Cell than HMT Buffer [0409 ] DOX - loaded thrombosomes were prepared by incubation with 600 uM DOX then following thrombosomes [0401 ] FIG . 12 shows a higher drug load per cell when manufacturing specifications . The concentration of DOX using loading buffer as compared to HMT buffer. The loaded into platelets was measured by fluorescence after amount of DOX is measured in mg/ cell by a fluorometer . sonication as described herein . FIG . 18 shows the concen tration of DOX loaded into platelets before and after Example 7 – DOX Induced Aggregation of Platelets lyophilization to a dry powder and rehydration was found to [0402 ] Loading platelets with DOX causes the platelets to be >43 % of the concentration measured immediately prior to aggregate resulting in a decrease in platelet count. The lyophilization . extent of platelet aggregation is measured as a function of [0410 ] FIG . 19 represents the same data from FIG . 18 , but the transmittance of light through a stirred suspension of the data were converted to molar concentration by using the platelets on an AggRAM . Platelets as single cells in a mean platelet volume (MPV ) after DOX loading . FIG . 19 suspension are too turbid for light to effectively pass shows that the intracellular DOX concentration is increased through . However , when platelets aggregate they fall out of 50 - fold prior to lyophilization and maintained at 30 - fold suspension and allow more light to pass through , thus after lyophilization relative to the external DOX concentra increasing the transmittance . FIG . 13A shows the DOX tion during incubation . induced platelet aggregation measured by percent light [0411 ] FIG . 20 shows that platelet counts , as measured by transmittance . the AcT -Diff hematology analyzer , remain mostly [0403 ] Platelets were treated with 0.36 mg/ ml, 0.72 unchanged after lyophilization . The target platelet count mg/ mL , 1 mg/ mL , and 3 mg/ml of DOX . Thrombin was when preparing the platelet suspensions for lyophilization used as a positive control. HMT and 1 mM magnesium was was 2000x10 ° /uL , and should remain similar after used as a negative control (FIG . 13A ) . lyophilization and rehydration in the same volume used to [0404 ] The concentration of platelets ( count ) is deter aliquot the platelet suspensions prior to lyophilization . mined on an AcT- Diff hematology analyzer . As shown in [0412 ] FIG . 21 shows that DOX -loaded thrombosomes FIG . 13B -C , the platelet count decreases with increasing have similar platelet receptor biomarker expression relative DOX concentration after incubation for 3 hours . The only to unlabeled thrombosomes immediately prior to and after anti -platelet compounds that shows effective inhibition of lyophilization . CD41 ( left hand columns) , CD62 (middle DOX - induced aggregation in this assay were the GPIIb / IIIa columns ), and Annexin V ( right hand columns ) expression inhibitors GR144053 and Tirofiban (See Table 1 ) levels weremeasured using fluorescently - labeled antibodies [0405 ] FIG . 13B shows that a GPIIb / IIIa inhibitor GR for each receptor . The fluorescent labels were chosen such 144053 ( > 1.2 uM ) limited reduction of platelet count fol that DOX fluorescence would not interfere with the antibody lowing coincubation with 0.5 mg/ ml DOX for 1 hour at 37° fluorescence signal. C. The data were generated from platelets pooled from seven [ 0413 ] FIG . 22 shows the batch containing Sublot A with apheresis units. Platelets (600,000 cells /uL ) , DOX ( 0 mg/ml 98 vials of unloaded thrombosomes and Sublot B with 98 top line, 0.5 mg/ml middle line, 1 mg/ ml bottom line ) , and vials of DOX - loaded thrombosomes adhere to collagen and GR 144053 ( 0 , 0.4 , 1.2 , 3.6 , 10.8 , and 32.4 uM . Tocris tissue factor coated microchips. Specifically , DOX -loaded Bioscience Cat. # 1263. ) were incubated in loading buffer for thrombosomes adhere to collagen and tissue factor coated 1 hour at 37° C. Following incubation , platelets were microchips similar to unloaded thrombosomes . The occlu quantified via Coulter AcoT diff2 Hematology Analyzer sion times were determined on a T - TAS by measurement of (x103 /UL ) . the pressure while suspensions of thrombosomes were flowed over coated surfaces . Example 8 – Platelets Retain DOX after [0414 ] FIG . 23 shows that DOX - loaded thrombosomes Cryopreservation or Lyophilization generate thrombin similar to unloaded thrombosomes. [ 0406 ] The concentration of Dox loaded into platelets Thrombin generation was determined by the measurement remains the same after cryopreservation . Platelets were of fluorescent signal from a substrate that does not fluoresce incubated with 600 UM Dox in the presence of PGE1 and until cleavage by thrombin , relative to a control. Cephalin is GR144053 at 37 ° C. for 3 hours , then washed , and subjected a positive control to show the maximum possible thrombin to cryopreservation in loading buffer containing 6 % poly generation in the assay, and octaplas is negative control sucrose . unable to generate thrombin . (0407 ] After cryopreservation , DOX can be released from [0415 ) FIG . 24 shows that DOX loaded thrombosomes within platelets in response to strong platelet activating can inhibit cancer cell growth more effectively than DOX agents described herein . The released DOX concentration alone . Dog hemangiosarcorcoma cells (DHSA ) (Kerafast , was measured by fluorescence on a plate reader after 2 ( left DHSA - 1426 , Cat. # EMN017 ) were plated at a fixed density hand columns) and 7 ( right hand columns ) days after incu in triplicate in 24 - well plates and grown overnight. The bation and centrifugation (FIG . 17 ). The released DOX is following day , the loaded and unloaded thrombosomes were shown here as a percentage of the total DOX measured rehydrated as described herein and assayed within one hour. before cryopreservation . The bottom line marks the DOX The DHSA cells were exposed to medium without throm release without agonist at 2 days and the top line marks the bosomes (HAS ) as a negative control and 1 % , 5 % , or 10 % DOX release without agonist after 7 days . unloaded thrombosomes and 1 % , 5 % , or 10 % DOX loaded US 2020/0206143 A1 Jul . 2 , 2020 27 thrombosomes, or free DOX ( in medium , 5 uM ). The next washed to remove excess fluorophore and fixed on micro day after about 24 hours of exposure, the test medium was scope slides at 4 ° C. overnight. Images were collected on a removed by gentle aspiration , the plates were washed once fluorescence microscope at 100x magnification . FIG . 27 with fresh medium , and fresh medium was added . The point shows the internalization of Paclitaxel into platelets . at which fresh medium was added is considered time zero [0421 ] While the embodiments of the invention are ame (FIG . 24 ) . At day 1 ( 24 hours ), 3 ( 72 hours ) and 5 ( 120 nable to various modifications and alternative forms, spe hours ), a 24 well plate was harvested . The triplicate wells cific embodiments have been shown by way of example in were pooled and counted using the Nexcelom cell counter the drawings and are described in detail below . The inten Auto 2000 with AO /PI live /dead cell count. tion , however, is not to limit the invention to the particular embodiments described . On the contrary , the invention is Example 9 — Olaparib - Loaded Platelets intended to cover all modifications, equivalents , and alter [0416 ] Poly ADP ribosome polymerase (PARP ) inhibitors natives falling within the scope of the invention as defined are drugs used in the treatment of cancer. PARP inhibitors by the appended claims. interfere with a cell's ability to repair DNA breaks. Olaparib [0422 ] 1. A method of preparing drug -loaded platelets, (also known as AZD - 2281, MK - 7339, or Lynparza® ) is a comprising: PARP inhibitor. FIG . 14 shows that Olaparib loads into [0423 ] treating platelets with a drug and with a loading platelets in a dose -dependent manner. After incubation with buffer comprising a salt, a base, a loading agent, and increasing total concentrations of PARPi (Olaparib ) and a optionally at least one organic solvent, fluorescently labeled PARPi- FL , keeping the ratio at 20: 1, to form the drug - loaded platelets . platelets were analyzed by flow cytometry . PARPi and [0424 ] 2. A method of preparing drug - loaded platelets , PARP -FL were tested at the following concentrations: 10 comprising : UM PARPi- FL and 0.5 mM PARPI, 30 ?M PARPi- FL and [0425 ) a ) providing platelets ; 1.5 mM PARPI, 60 UM PARPi- FL and 3 mM PARPi, and [0426 ] and 120 UM PARPi- FL and 6 mM PARPI . The percentage of [0427 ] b ) treating the platelets with a drug and with a loaded PARPi- loaded platelets increases with increasing loading buffer comprising a salt , a base , a loading concentrations as measured by the mean fluorescence inten agent, and optionally at least one organic solvent sity . FIG . 15 shows the total drug load increase following [0428 ] to form the drug - loaded platelets . incubation of cells with increasing concentrations of olparib . [0429 ] 3. The method of any one of the preceding embodi The platelets were sonicated and the intracellular fluores ments , wherein the platelets are treated with the drug and cence signal was measured on a plate reader . with the buffer sequentially , in either order . [0417 ] Olaparib loaded platelets were verified by loading [0430 ] 4. A method of preparing drug -loaded platelets , microscopy . FIG . 16 shows a fluorescence image. Olaparib comprising : is localized within platelets . After incubation with 80 uM ( 0431] (1 ) treating platelets with a drug to form a first PARPi- FL at 37 ° C. for 3 hours , platelets were washed to composition ; and remove excess fluorophore and fixed on microscope slides at [0432 ] (2 ) treating the first composition with a buffer 4 ° C. overnight. Images were collected on a fluorescence comprising a salt, a base, a loading agent, and option microscope at 100x magnification . These images show the ally at least one organic solvent, to form the drug internalization of PARPi into platelets . loaded platelets . [0433 ] 5. A method of preparing drug- loaded platelets , Example 10Paclitaxel -Loaded Platelets comprising : [0418 ] Paclitaxel is a chemotherapy agent that interferes [0434 ] (1 ) treating the platelets with a buffer comprising with the normal function of during cell divi a salt , a base , a loading agent , and optionally at least sion . Paclitaxel is loaded into platelets in a dose - dependent one organic solvent to form a first composition ; and manner. FIGS. 25A and B show incubation of platelets with [ 0435 ] ( 2 ) treating the first composition with a drug , to increasing total concentrations of Paclitaxel and fluores form the drug - loaded platelets . cently labeled Paclitaxel- Oregon Green . The platelets were [ 0436 ] 6. The method of embodiment 1 or 2 , wherein the kept at a ratio of 50 :1 , unlabeled drug to labeled drug . platelets are treated with the drug and with the buffer Uptake of paclitaxel was measured by flow cytometry . concurrently Measurements were taken at 2 , 4 , and 6 hours . As time [0437 ] 7. A method of preparing drug -loaded platelets , increased from 0 to 6 hours the concentration increased as comprising: measured by MFI and percent loaded increases . [0438 ] treating the platelets with a drug in the presence [0419 ] FIG . 26A - C shows the concentration of platelets of a buffer comprising a salt , a base , a loading agent, (measured by count) is determined on an AcT -Diff hema and optionally at least one organic solvent to form the tology analyzer during incubation with Paclitaxel . The plate drug - loaded platelets . let count is maintained as a function of increasing initial [0439 ] 8. The method of any one of the preceding embodi platelet concentration in the presence of paclitaxel, irrespec ments , wherein the platelets are pooled from a plurality of tive of the DMSO concentration . Minimally 50 % of platelets donors prior to a treating step . ( platelet count) are retained after loading . [0440 ] 9. A method of preparing drug - loaded platelets [0420 ] Paclitaxel loaded platelets were verified by loading comprising microscopy. FIG . 27 shows brightfield and fluorescence [ 0441 ] A ) pooling platelets from a plurality of donors ; images , followed by an overlay image. Paclitaxel is local and ized within platelets . After incubation with 100 uM Pacli [0442 ] B ) treating the platelets from step (A ) with a taxel- Oregon Green at 37 ° C. for 2 hours, platelets were drug and with a loading buffer comprising a salt, a base , US 2020/0206143 A1 Jul . 2 , 2020 28

a loading agent, and optionally at least one organic [0465 ] 22. Drug - loaded platelets prepared by the method solvent, to form the drug - loaded platelets . of any one of the preceding embodiments . [0443 ] 10. A method of preparing drug -loaded platelets [0466 ] 23.Rehydrated drug -loaded platelets prepared by a comprising method comprising rehydrating the drug - loaded platelets of [0444 ] A ) pooling platelets from a plurality of donors ; c embodiment 22 . and [0467 ] 24. The method of any one of the preceding [0445 ] B ) embodiments , wherein the drug is modified with an imaging [0446 ] ( 1 ) treating the platelets from step ( A ) with a agent . drug to form a first composition ; and [0468 ] 25. The method of embodiment 24 , wherein the [0447 ] ( 2 ) treating the first composition with a buffer drug is modified with the imaging agent prior to treating comprising a salt, a base , a loading agent, and platelets with the drug . optionally at least one organic solvent, to form the [ 0469 ] 26. The method of any one of the preceding drug - loaded platelets . embodiments , wherein the platelets are further treated with [0448 ] 11. A method of preparing drug- loaded platelets an imaging agent, wherein the drug - loaded platelets are comprising loaded with the imaging agent. [0449 ] A ) pooling platelets from a plurality of donors; [ 0470 ] 27. The method of any one of the preceding and embodiments , wherein the method does not comprise treat [ 0450 ] B ) ing the platelets with an organic solvent. [ 0451] ( 1 ) treating the platelets from step ( A ) with a [0471 ] 28. The method of any one of embodiments 4, 5 , 10 buffer comprising a salt, a base , a loading agent, and or 11 , wherein the method does not comprise treating the optionally at least one organic solvent, to form a first first composition with an organic solvent. composition ; and [ 0472 ] 29. The method of any one of the preceding [0452 ] ( 2 ) treating the first composition with a drug embodiments , wherein the method comprises treating the to form the drug - loaded platelets . platelets with Prostaglandin E1 (PGEI ) or Prostacyclin . [0453 ] 12. A method of preparing drug -loaded platelets [0473 ] 30. The method of any one of embodiments 1 to 28 , comprising wherein the method does not comprise treating the platelets [0454 ] A ) pooling platelets from a plurality of donors ; with Prostaglandin E1 (PGE1 ) or Prostacyclin . and [0474 ] 31. The method of any one of the preceding [0455 ] B ) treating the platelets with a drug in the embodiments , wherein the method comprises treating the presence of a buffer comprising a salt, a base, a loading platelets with a chelating agent such as EGTA . agent, and optionally at least one organic solvent, to [0475 ] 32. Themethod of any one of embodiments 1 to 30 , form the drug -loaded platelets . wherein the method does not comprises treating the platelets [0456 ] 13. The method of any one of the preceding with a chelating agent such as EGTA . embodiments , wherein the loading agent is a monosaccha [0476 ] 33. The method of any one of embodiments 1 to 29 , ride or a disaccharide . wherein the method comprises treating the first composition [ 0457 ] 14. The method of any one of the preceding with Prostaglandin E1 ( PGE1) or Prostacyclin . embodiments ,wherein the loading agent is sucrose, maltose , [ 0477 ] 34. The method of any one of embodiments 1 to 28 trehalose , glucose , mannose , or xylose . or 30 , wherein the method does not comprise treating the [ 0458 ] 15. The method of any one of the preceding first composition with Prostaglandin E1 (PGE1 ) or Prosta embodiments , wherein the platelets are isolated prior to a cyclin . treating step . [0478 ] 35. The method of any one of embodiments 1 to 31, [ 0459 ] 16. The method of any one of the preceding 33 or 34 , wherein the method comprises treating the first embodiments , wherein the platelets are loaded with the drug composition with a chelating agent such as EGTA . in a period of time of 5 minutes to 48 hours. [0479 ] 36. The method of any one of embodiments 1 to 30 [ 0460 ] 17. The method of any one of the preceding or 32 to 34 ,wherein the method does not comprises treating embodiments , wherein the concentration of drug in the the first composition with a chelating agent such as EGTA . drug -loaded platelets is from about 1 nM to about 100 mM . [0480 ] 37. The method of any one of the preceding [0461 ] 18. The method of any one of the preceding embodiments , wherein the method further comprises treat embodiments , wherein the one or more organic solvents ing the drug - loaded platelets with an anti -aggregation agent. selected from the group consisting of ethanol, acetic acid , [0481 ] 38. The method of embodiment 37 , wherein the acetone, acetonitrile , dimethylformamide, dimethyl sulfox anti- aggregation agent is a GPIIB /IIIa inhibitor. ide , dioxane, methanol, n - propanol, isopropanol, tetrahydro [0482 ] 39. The method of embodiment 38 , wherein the furan (THF ) , N -methyl pyrrolidone , dimethylacetamide GPIIB / IIIa inhibitor is GR144053 . ( DMAC ), or combinations thereof. [0483 ] 40. The method of embodiment 39 , wherein [ 0462 ] 19. The method of any one of the preceding GR144053 is present in a concentration of at least 1.2 uM . embodiments , further comprising cold storing , cryopreserv [0484 ] 41. The method of any one of embodiments 38-41, ing , freeze - drying , thawing , rehydrating , and combinations wherein the platelets are treated with the anti- aggregation thereof the drug - loaded platelets . agent before being treated with the drug . [ 0463] 20. The method of embodiment 19 , wherein the [0485 ] 42. The method of any one of embodiments 38-41 , drying step comprises freeze - drying the drug - loaded plate wherein the platelets are treated with the anti- aggregation lets . agent concurrently with the drug . [0464 ] 21. The method of embodiment 19 or 20 , further [0486 ] 43. The method of any one of the preceding claims, comprising rehydrating the drug - loaded platelets obtained wherein the drug is a small molecule , a protein , an oligo from the drying step . peptide , an aptamer , and combinations thereof. US 2020/0206143 A1 Jul . 2 , 2020 29

[ 0487 ] 44. The method of any one of the preceding 19. The method of claim 1 , further comprising cold embodiments , wherein the drug is a drug for the treatment storing , cryopreserving, freeze- drying , thawing, rehydrat of cancer. ing , and combinations thereof the drug - loaded platelets . [0488 ] 45. The method of embodiment 44 , wherein the 20. (canceled ) cancer comprises hemangiosarcoma. 21. ( canceled ) [ 0489 ] 46. The method of embodiments 44-45 , wherein 22. Drug - loaded platelets prepared by the method of the drug for the treatment of cancer is doxorubicin . claim 1 . [0490 ] 47. The method of embodiments 45-46 , wherein 23. (canceled ) the drug for the treatment of hemangiosarcoma is doxoru 24. The method of claim 1 , wherein the drug is modified bicin . with an imaging agent. [0491 ] 48. The method of embodiment 44 , wherein the drug for the treatment of cancer is paclitaxel . 25. ( canceled ) [0492 ] 49. The method of embodiment 44 , wherein the 26. (canceled ) drug for the treatment of cancer is a PARP inhibitor. 27. ( canceled ) [0493 ] 50. The method of embodiment 49 , wherein the 28. (canceled ) PARP inhibitor is olaparib . 29. The method of claim 1 ,wherein the method comprises 1. A method of preparing drug - loaded platelets , compris treating the platelets with Prostaglandin E1 (PGE1 ) , Pros ing : tacyclin , EGTA , or combinations thereof. treating platelets with a drug and with a loading buffer 30. ( canceled ) comprising a salt, a base, a loading agent, and option 31. ( canceled ) ally at least one organic solvent, 32. (canceled ) to form the drug - loaded platelets . 33. (canceled ) 2. (canceled ) 34. (canceled ) 3. The method of claim 1, wherein the platelets are treated 35. ( canceled ) with the drug and with the buffer concurrently or sequen 36. (canceled ) tially , in either order. 37. The method of claim 1 , wherein the method further 4. ( canceled ) comprises treating the drug - loaded platelets with an anti 5. (canceled ) aggregation agent. 6. (canceled ) 38. The method of claim 37 , wherein the anti -aggregation 7. (canceled ) agent is a GPIIb / IIIa inhibitor such as GR144053 . 8. The method of claim 1 , wherein the platelets are pooled 39. (canceled ) from a plurality of donors prior to a treating step . 40. ( canceled ) 9. A method of preparing drug- loaded platelets compris 41. The method of claim 37 , wherein the platelets are ing A ) pooling platelets from a plurality of donors ; and treated with the anti- aggregation agent before or concur B ) treating the platelets from step ( A ) with a drug and rently with the drug . with a loading buffer comprising a salt, a base , a 42. ( canceled ) loading agent, and optionally at least one organic 43. The method of claim 1 , wherein the drug is a small solvent, to form the drug - loaded platelets . molecule , a protein , an oligopeptide, an aptamer , and com 10. (canceled ) binations thereof . 11. ( canceled ) 44. The method of claim 1 , wherein the drug is a drug for 12. (canceled ) the treatment of cancer. 13. The method of any claim 1 , wherein the loading agent 45. The method of claim 44 , wherein the cancer comprises is a monosaccharide or a disaccharide . hemangiosarcoma. 14. The method of claim 1 , wherein the loading agent is 46. The method of claim 44 , wherein the drug for the sucrose, maltose , trehalose , glucose , mannose , or xylose . treatment of cancer is doxorubicin , paclitaxel , or a PARP 15. (canceled ) inhibitor. 16. (canceled ) 17. The method of any one of the preceding claims, 47. The method of claim 45 , wherein the drug for the wherein the concentration of drug in the drug - loaded plate treatment of hemangiosarcoma is doxorubicin . lets is from about 1 nM to about 100 mM . 48. (canceled ) 18. The method of claim 1 , wherein the one or more 49. ( canceled ) organic solvents selected from the group consisting of 50. The method of claim 469 , wherein the PARP inhibitor ethanol, acetic acid , acetone , acetonitrile , dimethylforma is olaparib . mide, dimethyl sulfoxide , dioxane, methanol, n - propanol, 51. The method of claim 37 , wherein the anti -aggregation isopropanol, tetrahydrofuran ( THF ), N -methyl pyrrolidone, agent is N -acetyl - cysteine . dimethylacetamide (DMAC ) , or combinations thereof.