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Publication Number
US-11447798-B2
Publication Date
2022-09-20
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 using a vector. A population of cells is sprayed with a volume of an aqueous solution that includes the payload and an alcohol at greater than 5 percent concentration.
The aqueous solution is formulated with less than 46 mM salt, less than 121 mM sugar, and less than 19 mM buffering agent. The salt is selected from NaCl, KCl, Na2HPO4, C2H3O2NH4, and KH2PO4, and dependent claim refinements further define alcohol identities and multi-ingredient formulations including H2O, ethanol, sucrose, potassium chloride, ammonium acetate, and HEPES.
The invention controls the delivered volume as a function of either the exposed surface area of the population of cells or a number of cells in the population of cells. Dependent claims further refine spraying by specifying gas-propelling the aqueous solution to form a spray and discrete liquid volume units with diameters in the range of 30 to 100 µm.
Claims Coverage
The document includes one independent claim that covers alcohol-containing, low-salt, low-sugar, low-buffer aqueous spraying for payload delivery across a cell plasma membrane. Dependent claims refine delivery volume dependence, spray formation, spray unit diameter, specific formulation ingredient ranges, and narrower payload examples.
Alcohol-containing aqueous spraying for plasma membrane payload delivery
A method for delivering a payload across a plasma membrane of a cell by providing a population of cells and spraying the population of cells with a volume of aqueous solution including the payload and an alcohol at greater than 5 percent concentration.
Low-salt, low-sugar, low-buffer aqueous solution formulation
Spraying an aqueous solution having less than 46 mM salt, less than 121 mM sugar, and less than 19 mM buffering agent, where the salt is selected from NaCl, KCl, Na2HPO4, C2H3O2NH4, and KH2PO4.
Volume as a function of exposed surface area or number of cells
The method wherein the volume is a function of exposed surface area of the population of cells or a number of cells in the population of cells.
Across the independent claim, the core coverage is centered on spraying cells with a payload-containing aqueous solution with greater than 5 percent alcohol, formulated with constrained salt, sugar, and buffering agent levels, and with a delivered volume defined as a function of either exposed surface area or number of cells. Dependent refinements narrow spraying to gas-propelled spray formation with discrete droplet or unit diameters and further constrain formulation to multi-ingredient ranges such as H2O, ethanol, sucrose, potassium chloride, ammonium acetate, and HEPES.
Stated Advantages
Improved delivery/viability compared with pipetting/micropipette, as referenced in the document’s performance/toxicity rationale [procedural detail omitted for safety].
Control of reversible membrane permeabilization is highlighted by limiting salt, sugar, and buffering agent while using alcohol at greater than 5 percent concentration.
Documented Applications
Delivery of payloads including nucleic acids, proteins, dyes, and quantum dots to cells, with readouts such as fluorescence microscopy and flow cytometry [procedural detail omitted for safety].
Use with adherent and suspension cells, including cells such as A549, CHO, HEK, HUVEC, primary MSC, fibroblasts, U226, and Jurkat [procedural detail omitted for safety].
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