# US 11,299,698 B2 · Delivery of materials to anucleate cells > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,299,698 B2 - Document ID: US11299698B2 - Status: Granted - Kind: B2 - Application number: 15/865,901 - Filed: 2018-01-09 - Published: 2018-07-19 - Granted: 2022-04-12 - Earliest claimed: 2015-07-09 - Assignees: Massachusetts Institute of Technology - Applicants: Massachusetts Institute of Technology - Inventors: Armon R. Sharei; Klavs F. Jensen; James Robbins Abshire; Jacquin Clarence Niles - Web page: https://patentgrep.com/patent/US11299698B2 ## CPC - C12M 23/16: {Microfluidic devices; Capillary tubes (integrated microfluidic structures B01L3/5027; microreactors B01J19/0093)} - A61K 35/18: Erythrocytes (haemoglobin A61K38/42) - A61P 7/06: Antianaemics - A61P 33/06: Antimalarials - C12M 35/04: {Mechanical means, e.g. sonic waves, stretching forces, pressure or shear stimuli} - C12N 5/0641: {Erythrocytes} - C12N 5/0644: {Platelets; Megakaryocytes} - C12N 15/87: Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation - C12N 2521/00: Culture process characterised by the use of hydrostatic pressure, flow or shear forces - C12N 2527/00: Culture process characterised by the use of mechanical forces, e.g. strain, vibration - Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change - C12M: APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; {APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS} - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE ## Abstract The current subject matter includes methods, systems, articles, and techniques to deliver material to anucleate cells, such as red blood cells. Using a rapid deformation based microfluidic system, loading of red blood cells with macromolecules of different sizes has been shown. Although delivery to some mammalian cells, such as cancer cell lines and fibroblasts had been previously demonstrated using this technique, those designs were incompatible with RBCs that have dramatically different physical properties. Through the use of smaller constriction sizes, high speeds and different buffers successful delivery to red blood cells can be achieved. By enabling robust delivery to red blood cells in a simple, scalable manner, the current subject matter can be implemented in a diversity of applications that deliver material to study red blood cell diseases and/or use red blood cells as a therapeutic platform. Related apparatus, systems, techniques, and articles are also described. ## Claims ### Claim 1 A method for delivering a payload into a mammalian anucleate cell comprising: passing the anucleate cell suspended in a buffer through a microfluidic channel that includes a cell-deforming constriction, wherein the step of passing the anucleate cell through the constriction causes perturbations of the membrane of the anucleate cell large enough for the payload to pass through, wherein the constriction comprises a width, a length and a depth, wherein the width of the constriction is less than 4 micrometers; incubating the anucleate cell in a solution comprising the payload for a predetermined time before or after the anucleate cell passes through the constriction; and delivering the payload into the anucleate cell. ### Claim 2 (depends on 1) The method of claim 1, wherein said cell is one or more of red blood cells, reticulocytes, and platelets. ### Claim 3 (depends on 2) The method of claim 2, wherein said cell is a red blood cell. ### Claim 4 (depends on 1) The method of claim 1, wherein said cell is a healthy cell. ### Claim 5 (depends on 4) The method of claim 4, wherein said cell is a red blood cell. ### Claim 6 (depends on 1) The method of claim 1, wherein said cell is an infected cell or a diseased cell. ### Claim 7 (depends on 6) The method of claim 6, wherein said cell is a red blood cell. ### Claim 8 (depends on 1) The method of claim 1, wherein the cell suspended in a buffer includes unmodified blood. ### Claim 9 (depends on 1) The method of claim 1, wherein the width of the constriction is between 0.5 micrometer and 4 micrometers. ### Claim 10 (depends on 9) The method of claim 9, wherein the width of the constriction is between 1 micrometer and 3 micrometers. ### Claim 11 (depends on 9) The method of claim 9, wherein said cell is a red blood cell. ### Claim 12 (depends on 9) The method of claim 9, wherein the width of the constriction is between 2 micrometers and 3 micrometers. ### Claim 13 (depends on 12) The method of claim 12, wherein said cell is a red blood cell. ### Claim 14 (depends on 9) The method of claim 9, wherein the width of the constriction is less than the largest diameter of the cell. ### Claim 15 (depends on 9) The method of any one of claim 9, wherein the width of the constriction is about 20% to about 99% the largest diameter of the cell. ### Claim 16 (depends on 1) The method of claim 1, wherein said buffer is a hypotonic buffer that causes said cell to swell. ### Claim 17 (depends on 1) The method of claim 1, wherein said payload-containing solution comprises one or more of proteins, peptides, small molecules, nucleic acids, lipids, carbohydrates, macromolecules, vitamins, polymers, fluorescent dyes, fluorophores, carbon nanotubes, quantum dots, nanoparticles, dextran polymers, and steroids. ### Claim 18 (depends on 1) The method of claim 1, wherein a cross-section of the microfluidic channel is selected from the group consisting of circular, elliptical, an elongated slit, square, hexagonal, and triangular. ### Claim 19 (depends on 1) The method of claim 1, wherein incubating the cell in a payload-containing solution comprises incubating the cell for 0.0001 seconds to 60 minutes. ### Claim 20 (depends on 1) The method of claim 1, wherein said length of the constriction is 30 micrometers or less. ### Claim 21 (depends on 1) The method of claim 1, wherein said depth of the constriction is between 1 micrometer and 1 millimeter. ### Claim 22 (depends on 1) The method of claim 1, wherein the microfluidic channel comprises a plurality of constrictions. ### Claim 23 (depends on 22) The method of claim 22, wherein the plurality of constrictions is arranged in series and/or parallel. ### Claim 24 (depends on 23) The method of claim 23, wherein the plurality of constrictions is arranged in parallel. ### Claim 25 (depends on 1) The method of claim 1, wherein the length of the constriction is between 10 micrometers and 30 micrometers. ### Claim 26 (depends on 1) The method of claim 1, wherein the length of the constriction is between 10 micrometers and 20 micrometers. ### Claim 27 (depends on 1) The method of claim 1, wherein said payload includes one or more of atovaquone-proguanil, artemether/lumefantrine, quinine sulfate, mefloquine, hydroxychloroquine, quinidine, sulfadoxine/pyrimethamine, amodiaquine, sulfonamides, halofantrine, doxycycline, tetracycline, hydrea, acyclovir, ganciclovir, valacyclovir, or penciclovir.