# US 8,785,177 B2 · Methods for nano-mechanoporation

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- Number: US 8,785,177 B2
- Document ID: US8785177B2
- Status: Granted
- Kind: B2
- Application number: 13/669,233
- Filed: 2012-11-05
- Published: 2013-05-30
- Granted: 2014-07-22
- Earliest claimed: 2011-11-04
- Assignees: The Board of Trustees of the University of Illinois, a body corporate and politic of the State of Illinois
- Applicants: Min-Feng Yu; Kyungsuk Yum; Ning Wang
- Inventors: Min-Feng Yu; Kyungsuk Yum; Ning Wang
- Web page: https://patentgrep.com/patent/US8785177B2

## CPC

- C12N 5/0006: {Modification of the membrane of cells, e.g. cell decoration}
- C12N 15/89: using microinjection
- B82Y 5/00: Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- B82Y: SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES

## Abstract

Methods for creating a transient nanoscale opening in a cell membrane and methods for transporting a desired species through the nanoscale opening are provided. A nano-sized needle-like tip can be used to mechanically slice the cell membrane to create a transient, localized nanoscale slit. The nanoscale slit may be used for transferring exogenous molecules into a living cell.

## Claims

### Claim 1

A method for creating a transient nano scale opening in the membrane of a cell, the method comprising the steps of:
a. providing a probe and a cell, the probe having a longitudinal axis and a solid tip end, the diameter of the tip end being from 10 to 300 nm and the cell being immersed in an extracellular solution;
b. contacting the end of the tip with the outer surface of the cell membrane;
c. following step b), piercing through the membrane of the cell by moving the probe in a longitudinal direction until the end of the tip is located within the intracellular environment of the cell;
d. following step c), creating an opening in the membrane of the cell by inducing motion of the tip in at least two directions transverse to the longitudinal axis of the probe, the motion of the tip comprising
i) motion of the tip in a first direction transverse to the longitudinal axis of the probe for a first lateral displacement distance at a first average lateral speed; and
ii) motion of the tip in a second direction transverse to the longitudinal axis of the probe for a second lateral displacement distance at a second average lateral speed, the second direction being other than the first direction;
e. following step d), retracting the tip completely from the cell and allowing the cell membrane to reseal the created opening,
wherein the cell is substantially stationary during steps b) through e) and the longitudinal speed of the probe in step c) and the first and second average lateral speeds of the probe in step d) are greater than zero and less than 100 microns/sec.

### Claim 2 (depends on 1)

The method of claim 1, wherein the probe is connected to a positioning component and motion of the tip is induced by translating the positioning component.

### Claim 3 (depends on 2)

The method of claim 2 wherein in step d)i) the positioning component is translated a first positioning component lateral translation distance and in step d)ii) the positioning component is translated a second positioning component lateral translation distance and the first and second positioning component lateral translation distances are each from 100 nm to 5 micrometers.

### Claim 4 (depends on 1)

The method of claim 1 wherein the longitudinal displacement of the tip during step d) is less than 5% of the first lateral displacement distance and less than 5% of the second lateral displacement distance.

### Claim 5 (depends on 1)

The method of claim 1 wherein the first and second directions are 180° apart, the first and second lateral displacement distances are the same and steps d) and e) are repeated at least 4 times.

### Claim 6 (depends on 1)

The method of claim 1 wherein the total time of tip motion in step d) is from 10 seconds to 3 minutes.

### Claim 7 (depends on 1)

The method of claim 1 wherein the tip has a tapered end.

### Claim 8 (depends on 1)

The method of claim 1, wherein the probe comprises a nanorod or a multiwalled nanotube.

### Claim 9 (depends on 8)

The method of claim 8, wherein the nanotube or nanorod material is selected from a group of materials having Young's modulus values higher than 50 GPa.

### Claim 10 (depends on 1)

The method of claim 1, wherein the cell remains viable.

### Claim 11 (depends on 1)

The method of claim 1, wherein the cell lacks a cell wall.

### Claim 12

A method for transporting a desired chemical species or biological species through a transient nanoscale opening in the membrane of a cell, the method comprising the steps of:
a. providing a probe and a cell, the probe having a longitudinal axis and a tip end, the diameter of the tip end being from 50 to 300 nm, the cell being immersed in an extracellular solution and the desired chemical species or biological species being provided either in the extracellular solution or within the cell;
b. contacting the end of the tip with the outer surface of the cell membrane;
c. following step b), piercing through the membrane of the cell by moving the probe in a longitudinal direction until the end of the tip is located within the intracellular environment of the cell;
d. following step c), creating a permeable opening in the membrane of the cell by inducing motion of the tip in at least two directions transverse to the longitudinal axis of the probe, the motion of the tip comprising
i) motion of the tip in a first direction transverse to the longitudinal axis of the probe for a first lateral displacement distance at a first average displacement speed; and
ii) motion of the tip in a second direction transverse to the longitudinal axis of the probe for a second lateral displacement distance at a second lateral average displacement speed, the second direction being other than the first direction;
e. transporting the desired chemical species or biological species through the permeable opening;
f. following step e), retracting the tip from the cell and allowing the cell membrane to reseal the created opening;
wherein the cell is substantially stationary during steps b) through f) and both the longitudinal speed of the probe in step c) and the first and second average lateral speeds of the probe in step d) are greater than zero and less than 100 microns/sec.

### Claim 13 (depends on 12)

The method of claim 12, wherein the extracellular solution comprises exogenous molecules and the exogenous molecules diffuse into the cell in step e).

### Claim 14 (depends on 13)

The method of claim 13 wherein the size of the exogenous molecules is from 1000 Dalton to 1000 kilo-Dalton.

### Claim 15 (depends on 12)

The method of claim 12 wherein the extracellular solution comprises detectable tags and the detectable tags diffuse into the cell in step e).

### Claim 16 (depends on 15)

The method of claim 15 wherein the detectable tags comprise nanoparticles having a size from 2 nm to 250 nm.

### Claim 17 (depends on 16)

The method of claim 16 wherein the nanoparticles are quantum dots.

### Claim 18 (depends on 12)

The method of claim 12 wherein endogenous molecules diffuse from the intracellular environment into the extracellular solution.

### Claim 19 (depends on 12)

The method of claim 12, wherein the probe is connected to a positioning component and motion of the tip is induced by translating the positioning component.

### Claim 20 (depends on 12)

The method of claim 12 wherein in step d)i) the positioning component is translated a first positioning component lateral translation distance and in step d)ii) the positioning component is translated a second positioning component lateral translation distance and the first and second positioning component lateral translation distances are each from 100 nm to 5 micrometers.

### Claim 21 (depends on 12)

The method of claim 12 wherein the longitudinal displacement of the tip in step d) is less than 5% of the first lateral displacement distance and less than 5% of the second lateral displacement distance.

### Claim 22 (depends on 12)

The method of claim 12 wherein the first and second directions are 180° apart, the first and second lateral displacement distances are the same and steps d) and e) are repeated at least 4 times.

### Claim 23 (depends on 12)

The method of claim 12 wherein the total time of tip motion is from 10 seconds to 3 minutes.

### Claim 24 (depends on 12)

The method of claim 12, wherein the tip has a tapered tip end.

### Claim 25 (depends on 12)

The method of claim 12, wherein the probe comprises a nanorod or a multiwalled nanotube.

### Claim 26 (depends on 25)

The method of claim 25, wherein the nanotube or nanorod material is selected from a group of materials having Young's modulus values higher than 50 GPa.

### Claim 27 (depends on 12)

The method of claim 12, wherein the cell remains viable.

### Claim 28 (depends on 12)

The method of claim 12, wherein the cell lacks a cell wall.