Platelets as delivery agents
Provided herein are RNA agent-loaded platelets, methods of preparing RNA agent-loaded platelets, and methods of using RNA agent-loaded platelets. In some embodiments, methods of loading RNA agents into platelets include treating platelets with a RNA agent, a cationic transfection reagent, and a loading buffer that can include a salt, a base, a loading agent, and optionally at least one organic solvent.
1. A method of preparing RNA agent-loaded freeze-dried platelet derivatives, comprising:
(a) treating platelets with a RNA agent that is a siRNA and/or a miRNA, in the presence of a cationic transfection reagent, and a loading buffer comprising a salt, a base, and a loading agent comprising a monosaccharide and/or a disaccharide for a time period in the range of 20 minutes to 12 hours, and at a temperature in the range of 18-42° C., to form RNA agent-loaded platelets; and
(b) lyophilizing the RNA agent-loaded platelets to form the RNA agent-loaded freeze-dried platelet derivatives, wherein the RNA agent-loaded freeze-dried platelet derivatives upon rehydration retain at least 10% of the RNA agent.
2. The method of claim 1 , wherein the loading buffer comprises at least one organic solvent, and wherein the organic solvent is ethanol.
3. The method of claim 1 , wherein the disaccharide is selected from the group consisting of sucrose, maltose, and trehalose, and the monosaccharide is selected from the group consisting of glucose, mannose, and xylose.
4. The method of claim 1 , wherein the cationic transfection reagent is a cationic lipid transfection reagent.
5. The method of claim 1 , wherein the concentration of RNA agent in the RNA agent-loaded platelets is from about 0.1 nM to about 10 nM.
6. The method of claim 1 , wherein the loading buffer comprises an organic solvent, and wherein the organic solvent is selected from the group consisting of ethanol, acetic acid, acetone, acetonitrile, dimethylformamide, dimethyl sulfoxide, dioxane, methanol, n-propanol, isopropanol, tetrahydrofuran (THF), N-methyl pyrrolidone, dimethylacetamide (DMAC), and combinations thereof.
7. The method of claim 1 , wherein the RNA agent-loaded freeze-dried platelet derivatives are further rehydrated to form rehydrated platelet derivatives.
8. The method of claim 1 , wherein the RNA agent-loaded freeze-dried platelet derivatives have less than 10% crosslinking of platelet membranes via proteins and/or lipids present on the membranes.
9. The method of claim 1 , wherein the RNA agent-loaded freeze-dried platelet derivatives upon rehydration retain at least 20% of the RNA agent.
10. The method of claim 1 , wherein the loading agent comprises a disaccharide, and wherein the disaccharide is trehalose, and wherein the time period is in the range of 30 minutes to 6 hours.
11. The method of claim 10 , wherein the loading agent comprises trehalose at a concentration of 10 mM to 500 mM.
12. The method of claim 1 , wherein pH of the platelets prior to the treating step is in the range of 6.0 to 7.4.
13. The method of claim 1 , wherein treating the platelets comprises loading a concentration of the RNA agent from 0.1 nM to 10 μM to form the RNA agent-loaded platelets.
14. The method of claim 1 , wherein the method further comprises providing the platelets in the loading buffer before treating the platelets.
15. The method of claim 1 , wherein treating comprises mixing the RNA agent and the cationic transfection agent to form a mixture and treating the platelets with the mixture to form RNA agent-loaded platelets.
16. The method of claim 10 , wherein the method further comprises incubating the RNA agent-loaded platelets with polysucrose before the lyophilizing.
17. The method of claim 16 , wherein polysucrose is in the range of 3% to 7% w/v.
18. The method of claim 16 , wherein the RNA agent-loaded freeze-dried platelet derivatives are further rehydrated to form rehydrated platelet derivatives, and wherein the rehydrated platelet derivatives retain at least 90% of channel occlusion in a Total Thrombus-formation Analysis System (T-TAS).
19. method of claim 1 , wherein the RNA agent is siRNA, the loading agent comprises a disaccharide, and the disaccharide is trehalose.
20. The method of claim 10 , wherein the loading agent comprises trehalose at a concentration of 100 mM to 500 mM.
21. The method of claim 10 , wherein the loading agent comprises trehalose at a concentration of 100 mM to 150 mM.
22. The method of claim 1 , wherein the RNA agent-loaded freeze-dried platelet derivatives upon rehydration retain at least 30% of the RNA agent.