(483d) Microfluidic Cell Deformation As a Robust, Vector-Free Method for Cytosolic Delivery of Macromolecules

Introduction

Intracellular delivery of
macromolecules is a critical step in therapeutic and research applications.
Nanoparticle mediated delivery of DNA and RNA, for example, is being explored
for gene therapy¹,
while protein delivery is a promising means of affecting cellular function in
both clinical² and
laboratory³
settings. Other materials, such as small molecules, quantum dots, or gold
nanoparticles, are of interest for cancer therapies⁴, 
intracellular labeling⁵, and
single molecule tracking⁶.

The cell membrane is largely
impermeable to macromolecules. Many existing techniques utilize polymeric
nanoparticles⁷, or
chemical modifications of the target molecule, such as cell penetrating
peptides (CPPs) ⁸, to
facilitate membrane poration or endocytotic delivery. In these methods, the
delivery vehicle's efficacy is often dependent on the structure of the target
molecule and the cell type. These methods are thus efficient in the delivery of
structurally uniform materials, such as nucleic acids, but often ill-suited for
the delivery of more structurally diverse materials, such as proteins⁹ and
some nanomaterials¹⁰.
Moreover, the endosome escape mechanism that most of these methods rely on is
often inefficient; hence, much material remains trapped in endosomal and
lysosomal vesicles.

In this presentation, we
detail a method for cytosolic delivery based on rapid mechanical deformation of
the cell to produce transient membrane disruptions that facilitate the passive
diffusion of material into the cell cytosol.  This method was developed with
the aim of delivering almost any macromolecule of interest to almost any cell
type, at high throughput. Although shear based delivery methods have been
demonstrated previously, they are unsuitable for some applications due to low
viability and/or delivery efficiency¹¹. The
proposed technique, however, is distinct because it uses a physical
constriction to deform and shear the cells in a controlled, reproducible
manner. Unlike most current methods, this approach does not rely on electric
fields, exogenous materials, endocytosis or chemical modification of the target
molecule. Our data indicate that this method could be particularly advantageous
for applications involving nanomaterials, proteins, or difficult-to-transfect
cell types, such as immune cells and stem cells - all of which are often
underserved by current methods.

Delivery method

We hypothesize that the
rapid mechanical deformation of a cell, as it passes through a constriction
with a minimum dimension smaller than the cell diameter, results in the
formation of transient membrane disruptions or pores (Fig. 1a). The size
and frequency of these pores would be a function of the shear and compressive
forces experienced by the cell during its passage through the constriction. 
Material from the surrounding medium may then diffuse directly into the cell
cytosol throughout the lifespan of these pores. Such an approach could
theoretically enable the diffusive delivery of any macromolecule small enough
to fit through the pores. To implement this approach, we generated a family of
microfluidic devices with different constriction dimensions and numbers of
constrictions in series.  

Each device consists of 45
identical, parallel microfluidic channels, containing one or more
constrictions, etched onto a silicon chip and sealed by a Pyrex layer. The
width and length of each constriction range from 4-8um and 10-40um
respectively. The current design is typically operated at a throughput rate of
20,000 cells/s, yielding close to one million treated cells per device prior to
failure, due to clogging. The parallel channel design was chosen to increase
throughput, while insuring uniform treatment of cells, because any clogging or
defects in one channel cannot affect the flow speed in neighboring channels
(the device is operated at constant pressure). Prior to use, the device is
first connected to a steel interface that connects the inlet and outlet reservoirs
to the silicon device. A mixture of cells and the desired delivery material is
then placed into the inlet reservoir and Teflon tubing is attached at the inlet
(not shown). A pressure regulator is then used to adjust the pressure at the
inlet reservoir and drive the cells through the device. Treated cells are
collected from the outlet reservoir.

An enabling platform

In order to illustrate our
method's potential in addressing current delivery challenges, we conducted a
number of proof-of-concept experiments using various nanomaterials and proteins
of interest. Antibodies to tubulin, for example, were delivered (Fig. 1b)
using this technique, yielding a diffuse distribution throughout the cell that
would be consistent with cytosolic delivery. PEG1000 coated, 15nm gold
nanoparticles were also delivered as measured by tunneling electron microscopy
(TEM) of fixed cells (Fig. 1c). These nanoparticles appear to be mostly
un-aggregated and are not visibly sequestered into endosomes. We were also able
to verify the successful delivery of carbon nanotubes¹²
(encapsulated by a DNA oligonucleotide) by flow cytometry and Raman
spectroscopy. The aforementioned materials are currently difficult to deliver
to the cell cytosol and each material often requires a specialized modification
to facilitate delivery. In our work, all four materials were delivered to HeLa
cells using the same set of conditions on a 10um-6umx5 device. In addition,
this method has demonstrated the potential to accommodate a diverse range of
cell types, including embryonic stem cells and primary lymphocytes (Fig.
1d,e), thereby signaling its potential for use across a range of research
and perhaps clinical applications.

Figure 1. An overview of the
delivery system and its capabilities. a) Illustration of delivery
hypothesis whereby the rapid deformation of a cell, as it passes through a
microfluidic constriction, generates transient membrane pores. Includes an
electron micrograph of current parallel channel design with blue cells as an
illustration. b) Fluorescent micrographs of HeLa cells immediately after
delivery of antibodies to tubulin with an Alexa Fluor 488 tag. Scale bars at
5um. c) Tunneling electron microscopy (TEM) images of gold nanoparticles (some
indicated by arrows) in a cell fixed ~1s after treatment. Scale bar at 500nm.
d) Delivery of pacific blue labeled 3kDa dextran and e) fluorescein labeled
70kDa dextran to Macrophages (CD11b+), B lymphocytes (CD19+) and T lymphocytes
(TCR+) derived from the blood of BL6 mice. The 30um-5umx5 and 30um-5um
conditions denote two different device designs.

Summary

We have detailed a new
method for cytosolic delivery that relies on the rapid mechanical deformation
of a cell to induce transient membrane poration. This technique has
demonstrated the potential to deliver a broad range of materials, some of which
are challenging to use with current methods, to a variety of
difficult-to-transfect cell types, including stem cells and immune cells. By
providing such flexibility in application and obviating the need for exogenous
materials or electrical fields, this method could potentially enable new
avenues of disease research and treatment. Indeed our work has demonstrated
this systems ability to deliver carbon nanotubes and antibodies to live cells ?
applications that could enable new sensing and imaging modalities.

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