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Publication Number
US-10612042-B2
Publication Date
2020-04-07
Expiration Date
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.
Core Innovation
The invention provides a method for delivering a payload across a plasma membrane of a cell without a viral vector. A population of cells is contacted with a volume of an aqueous solution that includes the payload and an alcohol at greater than 5 percent concentration, and the contacting performs intracellular delivery of the payload while maintaining cell viability and supports reversible permeabilisation of the plasma membrane.
The aqueous solution is delivered by gas propelling the aqueous solution to form a spray. The spray provides transient exposure and an evenly distributed distribution of exposure to the cells, and the volume of the aqueous solution is determined as a function of exposed surface area of the population of cells and/or a number of cells in the population of cells.
The method further includes aqueous solution compositions that constrain alcohol concentration and, where described, buffer components and osmolarity/pH to support delivery and viability. The delivered payload can include nucleic acids, including small interfering RNA, microRNA, and plasmid DNA; peptides/proteins and protein examples; small chemical molecules; and antibodies or antibody fragments, including co-delivery payloads where described.
Claims Coverage
The patent document contains one independent claim. The independent claim covers a gas-propelled spray method for intracellular delivery across a plasma membrane using an aqueous payload solution with alcohol at greater than 5 percent concentration, with spray volume determined by exposed surface area and/or cell number. Dependent claims refine the method with additional quantitative and compositional limitations and constrain allowable payload classes and cell types.
Alcohol-containing aqueous payload for plasma membrane delivery
Contacting a population of cells with a volume of aqueous solution including the payload and an alcohol at greater than 5 percent concentration to deliver the payload across the plasma membrane.
Gas-propelled spraying and volume determined by exposed surface area and/or cell number
Performing contacting by gas propelling the aqueous solution to form a spray, wherein the volume is a function of exposed surface area of the population of cells and/or a number of cells in the population of cells.
Spray discrete units and specified propelling gas
Spray discrete units having diameters of 30-100 μm, wherein the gas is nitrogen, ambient air, or an inert gas.
Alcohol concentration range in the aqueous solution
Defining an ethanol concentration in the aqueous solution of 5% to 30%.
Volume per cell or per exposed surface area constraints
Specifying the aqueous-solution volume using ranges or approximate values expressed per cell or per square micrometer of exposed surface area, with “about” constrained within 10%.
Payload molecular-mass/size constraints and listed payload examples
Limiting payloads such as small chemical molecules, peptide/protein, and nucleic acids by molecular-mass/size constraints and/or listed examples.
Allowed cell populations including adherent and non-adherent cells
Defining the population of cells as adherent cells and/or non-adherent cells, including optional specified subtypes.
Overall, the claim set focuses on intracellular delivery across a plasma membrane using an aqueous payload solution with alcohol at greater than 5 percent concentration, delivered by gas-propelled spraying. Coverage is further refined by dependent limitations on spray characteristics, alcohol/ethanol concentration, spray/contact volume tied to exposed surface area or cell number, constrained payload types and molecular-mass/size ranges, and specified adherent/non-adherent cell populations.
Stated Advantages
Improved intracellular delivery of payloads while maintaining viability.
Reduced toxicity compared with pipetting.
Documented Applications
Delivering intracellular payloads including siRNA, dextrans, proteins, mRNA, plasmid DNA, and quantum dots using gas-propelled atomized spray.
Cell types described as used for delivery include A549 cells, CHO cells, primary fibroblasts, mesenchymal stem cells, U226 suspension cells, and Jurkat cells.
Using payload examples for intracellular detection/assays including GFP mRNA and luciferase mRNA, with cell viability and intracellular uptake assessment.
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