# US 11,999,966 B2 · Apparatuses and methods using nanostraws to deliver biologically relevant cargo into non-adherent cells

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- Number: US 11,999,966 B2
- Document ID: US11999966B2
- Status: Granted
- Kind: B2
- Application number: 17/081,983
- Filed: 2020-10-27
- Published: 2021-03-04
- Granted: 2024-06-04
- Earliest claimed: 2017-07-19
- Assignees: The Board of Trustees of the Leland Stanford Junior University
- Applicants: The Board of Trustees of the Leland Stanford Junior University
- Inventors: Karl Martin Hjort; Sergio Leal-Ortiz; Yuhong Cao; Chris Rehse; Andy Kah Ping Tay; Nicholas A. Melosh
- Web page: https://patentgrep.com/patent/US11999966B2

## CPC

- C12N 15/87: Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12M 23/02: {Form or structure of the vessel (large containers B65D88/00)}
- C12M 23/42: {Integrated assemblies, e.g. cassettes or cartridges}
- C12M 35/00: {Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion (machines for extracting juice from animal or plant tissue by electroplasmolysis A23N1/006, processes employing electric or wave energy B01J19/08; treatment of microorganisms or enzymes with electrical or wave energy C12N13/00; methods for cell fusion C12N15/02; introduction of foreign genetic material C12N15/87)}
- C12M 35/02: {Electrical or electromagnetic means, e.g. for electroporation or for cell fusion}
- C12M 35/04: {Mechanical means, e.g. sonic waves, stretching forces, pressure or shear stimuli}
- B82Y 5/00: Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
- B82Y 40/00: Manufacture or treatment of nanostructures
- C12M 23/16: {Microfluidic devices; Capillary tubes (integrated microfluidic structures B01L3/5027; microreactors B01J19/0093)}
- C12N 5/0068: {General culture methods using substrates (for specific animal cell type C12N5/06)}
- C12N 5/0075: {using microcarriers}
- C12N 2521/00: Culture process characterised by the use of hydrostatic pressure, flow or shear forces
- C12N 2535/00: Supports or coatings for cell culture characterised by topography
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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}
- B82Y: SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES

## Abstract

Nanostraws and to methods of utilizing them in order to deliver biologically relevant molecules such as DNA, RNA, proteins etc., into non-adherent cells such as immune cells, embryos, plant cells, bacteria, yeast etc. The methods described herein are repeatedly capable of delivering biologically relevant cargo into non-adherent cells, with high cell viability, dosage control, unaffected proliferation or cellular development, and with high efficiency. Among other uses, these new delivery methods will allow to scale pre-clinical cell reprogramming techniques to clinical applications.

## Claims

### Claim 1

A flow-through apparatus for delivering a biologically relevant cargo into non-adherent cells, the apparatus comprising:
a first flow path having an inlet port and an outlet port, wherein the first flow path is configured to pass a suspension of cells from the inlet port to the outlet port;
a deflectable substrate along a lateral side of the first flow path;
a plurality of nanostraws extending through the deflectable substrate and into the first flow path, wherein the plurality of nanostraws extend from the flexible substrate, further wherein the plurality of nanostraws are hollow and have an inner diameter of between 5 nm-1500 nm;
a reservoir in fluid communication with an inside of the plurality of nanostraws, the reservoir configured to hold the biologically relevant cargo;
wherein the deflectable substrate is configured to deflect between a first position and a second position, further wherein the plurality of nanostraws extend further into the first flow path in the second position than in the first position to at least partially occlude the first flow path so that non-adherent cell in the flow path communicate with one or more of the plurality of nanostraws.

### Claim 2 (depends on 1)

The apparatus of claim 1, further comprising a pair of electrodes on opposite sides of the first flow path configured to apply an electrical field between the reservoir and the first flow path.

### Claim 3 (depends on 1)

The apparatus of claim 1, further comprising a pair of electrodes on opposite sides of the first flow path configured to apply an electrical field between the reservoir and the first flow path when the deflectable substrate is in the second position.

### Claim 4 (depends on 1)

The apparatus of claim 1, wherein the reservoir is configured as a second fluidic channel through which the biologically relevant cargo flows.

### Claim 5 (depends on 1)

The apparatus of claim 1, wherein the first flow path comprises an inlet at a first end and an outlet at a second end.

### Claim 6 (depends on 1)

The apparatus of claim 1, wherein the deflectable substrate comprises a porous structure.

### Claim 7 (depends on 1)

The apparatus of claim 1, wherein the plurality of nanostraws extend between 2 nm and 50 μm from the substrate.

### Claim 8 (depends on 1)

The apparatus of claim 1, wherein the deflectable substrate is configured to transition from the first position to the second position based on the pressure in the reservoir.

### Claim 9 (depends on 1)

The apparatus of claim 1, wherein the apparatus is configured as a cartridge.

### Claim 10

An apparatus for delivering a biologically relevant cargo into non-adherent cells, the apparatus comprising:
a first flow path having an inlet port and an outlet port, wherein the first flow path is configured to pass a suspension of cells from the inlet port to the outlet port;
a deflectable substrate along a lateral side of the first flow path;
a plurality of nanostraws extending through the deflectable substrate and into the first flow path, wherein the plurality of nanostraws extend between 2 nm and 50 μm from the substrate, further wherein the plurality of nanostraws are hollow and have an inner diameter of between 5 nm-1500 nm;
a reservoir in fluid communication with an inside of the plurality of nanostraws, the reservoir configured to hold the biologically relevant cargo;
wherein the deflectable substrate is configured to deflect between a first position and a second position, further wherein the plurality of nanostraws extend further into the first flow path in the second position than in the first position to at least partially occlude the first flow path so that non-adherent cell in the flow path communicate with one or more of the plurality of nanostraws; and
a pair of electrodes on opposite sides of the first flow path configured to apply an electrical field between the reservoir and the first flow path.