Claims for Patent: 10,612,042

Summary for Patent: 10,612,042

Title:	Delivery across cell plasma membranes
Abstract:	Delivering a payload across a plasma membrane of a cell includes providing a population of cells and contacting the population of cells with a volume of an aqueous solution. The aqueous solution includes the payload and alcohol content greater than 5 percent concentration. The volume of the aqueous solution may be a function of exposed surface area of the population of cells, or may be a function of a number of cells in the population of cells. Related compositions, apparatus, systems, techniques, and articles are also described.
Inventor(s):	Maguire; Michael (Dublin, IE), O\'Dea; Shirley (Maynooth, IE)
Assignee:	Avectas Limited (Kildare, IE)
Application Number:	15/521,192
Patent Claims:	1. A method for delivering a payload across a plasma membrane of a cell, comprising: providing a population of cells; and contacting the population of cells with a volume of aqueous solution, the aqueous solution including the payload and an alcohol at greater than 5 percent concentration, wherein the volume is a function of: (i) exposed surface area of the population of cells; or (ii) a number of cells in the population of cells, and wherein contacting the population of cells with the volume of aqueous solution is performed by gas propelling the aqueous solution to form a spray. 2. The method of claim 1, wherein the volume is (a) between 6.0.times.10.sup.-7 microliter per cell and 7.4.times.10.sup.-4 microliter per cell; (b) between 9.3.times.10.sup.-6 microliter per cell and 2.8.times.10.sup.-5 microliter per cell; (c) between 4.9.times.10.sup.-6 microliter per cell and 2.2.times.10.sup.-3 microliter per cell; (d) about 1.9.times.10.sup.-5 microliters per cell, wherein about is within 10 percent; (e) between 2.6.times.10.sup.-9 microliter per square micrometer of exposed surface area and 1.1.times.10.sup.-6 microliter per square micrometer of exposed surface area; (f) between 5.3.times.10.sup.-8 microliter per square micrometer of exposed surface area and 1.6.times.10.sup.-7 microliter per square micrometer of exposed surface area; or (g) about 1.1.times.10.sup.-7 microliter per square micrometer of exposed surface area, wherein about is within 10 percent. 3. The method of claim 2, wherein the spray comprises a colloidal or sub-particle comprising a diameter of 1 nm to 100 .mu.m. 4. The method of claim 3, wherein the particle comprises a diameter of 30 to 100 .mu.m. 5. The method of claim 1, wherein (a) said spray comprises discrete units of volume ranging in size from 30-100 .mu.m in diameter; (b) said spray comprises discrete units of volume with a diameter of about 30-50 .mu.m, wherein about is within 10 percent; or (c) said gas comprises nitrogen, ambient air, or an inert gas. 6. The method of claim 1, wherein (a) a total volume of aqueous solution of 20 .mu.l is delivered to a cell-occupied area of about 1.9 cm.sup.2, wherein about is within 10 percent; or (b) a total volume of aqueous solution of 10 .mu.l is delivered to a cell-occupied area of about 0.95 cm.sup.2, wherein about is within 10 percent. 7. The method of claim 1, wherein (a) the population of cells is in contact with said aqueous solution for 0.1-10 minutes prior to adding a second volume of buffer or culture medium to submerse or suspend said population of cells; (b) the population of cells is in contact with a spray for about 1-2 minutes prior to adding the second volume of buffer or culture medium to submerse or suspend the population of cells; (c) the population of cells is in contact with the aqueous solution for 2 seconds to 5 minutes prior to adding a second volume of buffer or culture medium to submerse or suspend the population of cells; or (d) the population of cells is in contact with the aqueous solution for 30 seconds to 2 minutes prior to adding a second volume of buffer or culture medium to submerse or suspend the population of cells. 8. The method of claim 7, wherein the buffer or culture medium comprises phosphate buffered saline (PBS). 9. The method of claim 1, wherein said aqueous solution comprises an ethanol concentration of 5 to 30%. 10. The method of claim 1, wherein said aqueous solution comprises one or more of 75 to 98% H.sub.2O, 2 to 45% ethanol, 6 to 91 mM sucrose, 2 to 35 mM potassium chloride, 2 to 35 mM ammonium acetate, and 1 to 14 mM (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES). 11. The method of claim 1, wherein (a) the population of cells comprises adherent cells; (b) the population of cells comprises adherent cells, and wherein the adherent cells include at least one of primary or immortalized mesenchymal stem cells, lung cells, neuronal cells, fibroblasts, human umbilical vein (HUVEC) cells, and human embryonic kidney (HEK) cells; (c) the population of cells comprises non-adherent cells; or (d) the population of cells comprises non-adherent cells, and wherein the non-adherent cells include at least one of primary or immortalized hematopoietic stem cell (HSC), T cells, natural killer (NK) cells, cytokine-induced killer (CIK) cells, human cord blood CD34+ cells, B cells. 12. The method of claim 1, wherein the payload comprises a small chemical molecule, a peptide or protein, or a nucleic acid. 13. The method of claim 12, wherein (a) the small chemical molecule comprises a molecular mass of less than 1,000 Da; (b) the small chemical molecule comprises a molecular mass of less than 1,000 Da, and wherein the small chemical molecule comprises MitoTracker.RTM. Red CMXRos, propidium iodide, methotrexate, or DAPI (4',6-diamidino-2-phenylindole); (c) the peptide comprises ecallantide, liraglutide, or icatibant; (d) the nucleic acid comprises a small-interfering RNA (siRNA); (e) the nucleic acid comprises a small-interfering RNA (siRNA), and wherein the siRNA molecule comprises a molecular mass of about 15,000 Da; (f) the protein comprises a molecular mass about 1,000-150,000 Da; (g) the protein comprises an antibody, or fragment thereof; (h) the protein comprises an antibody, or fragment thereof, and wherein the antibody or fragment thereof comprises an anti-actin antibody, an anti-GAPDH antibody, an anti-Src antibody, an anti-Myc ab, and an anti-Raf antibody; or (i) the nucleic acid molecule comprises greater than 5,000,000 Da. 14. The method of claim 1, wherein the payload comprises a therapeutic agent, a diagnostic agent, a fluorescent molecule, or a detectable nanoparticle. 15. The method of claim 14, wherein (a) the therapeutic agent includes at least one of cisplatin, aspirin, a statin, and fluoxetine; (b) the diagnostic agent includes at least one of methylene blue, patent blue V, and indocyanine green; or (c) the nanoparticle comprises a quantum dot. 16. The method of claim 1, wherein the population of cells is substantially confluent, wherein substantially is greater than 75 percent confluent. 17. The method of claim 1, wherein the population of cells form a monolayer of cells. 18. The method of claim 1, wherein the population of cells comprises adherent cells, and wherein the adherent cells comprise lung cells. 19. The method of claim 1, wherein the population of cells comprises A549 cells. 20. The method of claim 1, wherein the population of cells comprises macrophages. 21. The method of claim 1, wherein the population of cells comprises mesenchymal stem cells. 22. The method of claim 1, wherein the payload comprises nucleic acid. 23. The method of claim 1, wherein the payload comprises mRNA.

Details for Patent 10,612,042

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Takeda Pharmaceuticals U.s.a., Inc.	KALBITOR	ecallantide	Injection	125277	12/01/2009		2034-10-24