TECHNICAL FIELD
The present disclosure relates to cell manipulation. Moreover, the present
disclosure relates to a method of selectively manipulating cells using magnetic
nanostructures.
5 BACKGROUND
Interaction of nanostructures (or nanomotors) with living cells is of fundamental
importance. Intracellular sensing (i.e., detecting specific conditions or biomarkers
within a cell) and delivery of payload (delivering therapeutic or diagnostic agents
to that cell) are witnessing immense development in recent days. Conventionally,
10 fluorescent molecules or biosensors (such as protein-based sensors) can be designed
to bind or detect specific targets (or condition) within cells to visualize and quantify
the presence of certain biomolecules therein. Once the specific targets (or
condition) are detected, a payload (such as drugs, genes, or nanoparticles) may be
to the target cell for therapeutic or diagnostic purposes, using conventional methods
15 such as nanoparticles (through passive diffusion or active targeting), liposomes,
viral vectors, cell-penetrating peptides, and so on.
Advancements in nanotechnology, bioengineering, and molecular biology continue
to drive progress in intracellular sensing and delivery, offering new possibilities for
precision medicine and personalized therapies. In this regard, especially,
20 nanostructure-cell interaction is gaining global interest. Out of the different
available modalities of nanostructure-cell interaction, cell sorting and manipulation
(namely, magnetic manipulation) is unique as it can manoeuvre nanostructures
inside cells with great precision and has been used for mechanical measurements of
intracellular environments. In recent years, cell sorting has been performed using
25 both non-magnetic and magnetic forcing tools (or strategies). Non-magnetic tools
include microfluidic flow engineering, optical tweezers, dielectrophoresis, acoustic
waves, and fluorescence activated cell sorting (FACS). Magnetic tools are typically
based on magnetic beads that bind to specific cell surface markers and which can
be manipulated by external magnetic fields. For example, magnetic-activated cell
30 sorting (MACS) uses a magnetized column to capture cells that are labelled with
3
magnetic beads. Magnetic tools are particularly attractive due to the benign nature
of bio-magnetic interactions, as well as their high purity, low cost, and minimal
damage to the cells. Specifically, nanostructures such as nanorobots (or nanobots
or nanomotors) are tiny machines that can manoeuvre through a medium to achieve
5 a predefined task such as payload delivery and local sensing. However,
conventional nanostructures fail to achieve different functionalities from the same
class of nanostructures resulting in the possibility of non-specific binding, and the
difficulty of separating cells with low expression levels of the target antigen, and
so on. Moreover, conventional nanostructures also fail to decouple cellular
10 manipulation and intracellular.
Therefore, in light of the foregoing discussion, there exists a need to overcome the
aforementioned drawbacks associated with the conventional method for
mobilization of the entities from a sensitive substrate.
SUMMARY
15 The present disclosure provides a method of selectively manipulating cells using
magnetic nanostructures. The present disclosure provides a solution to the technical
problem of how to effectively sort and move cells with a medium. An aim of the
present disclosure is to provide a solution that overcomes at least partially the
problems encountered in the prior art and provide selectively manipulating cells
20 using magnetic nanostructures within the cells in a remote manner using magnetic
field, electric field and acoustic energy or some combinations of those forces. These
cells with magnetic nanostructures inside them can then be used to interact with
non-magnetic cells in their surroundings to achieve active manipulation of a swarm
of cells.
25 One or more objectives of the present disclosure is achieved by the solutions
provided in the enclosed independent claims. Advantageous implementations of the
present disclosure are further defined in the dependent claims.
In one aspect, the present disclosure provides a method of selectively manipulating
cells using magnetic nanostructures. The method comprises incubating live cells
4
with the magnetic nanostructures to obtain magnetic nanostructures-internalized
cells; and remotely activating the magnetic nanostructures-internalized cells for cell
manipulation by applying an external force, wherein the activated magnetic
nanostructures-internalized cells interact with surrounding non-active non5 magnetic cells circulating in a fluid medium to achieve active manipulation of a
swarm of cells, and wherein the swarm of cells comprises the activated magnetic
nanostructures-internalized cells and the non-active non-magnetic cells.
The method of the present disclosure introduces nanostructures for effective cell
manipulation of a swarm of cells. The active manipulation of a swarm of cells
10 comprising magnetic and non-magnetic cells from a remote location facilitates a
nuanced approach providing unique opportunities for cell level interrogation and
manipulation. Specifically, the use of helical-shaped magnetic nanostructures
provides excellent manoeuvrability inside living cells and thereafter, altering the
interaction between cell and substrate (namely, walls of the vessel containing the
15 fluid medium), thereby demonstrating active manipulation of the cells. Moreover,
the disclosed method also discusses cell manipulation with fluidic forces,
understood through detailed numerical simulations of the hydrodynamic
interactions. Beneficially, the method enables integrating cell-level biophysical
measurements with sorting and active manipulation of cell using the same class of
20 magnetic nanostructures (nanomotors or nanobots). In this regard, the same class
of magnetic nanostructures are tuned to facilitate interaction between cell and the
substrate, to establish the efficacy and potential of the method and the magnetic
nanostructures for future biomedical industry. The magnetic nanostructures are
made to manoeuvre inside living cells with different cell-substrate interactions by
25 application of rotating magnetic field. Moreover, such magnetic nanostructures are
designed to work as non-contact cell manipulation agents for controllably
manipulating other passive cells which do not have internalized magnetic
nanostructures, via hydrodynamic interactions. Additionally, beneficially, the
method enables decoupling of the cellular manipulation and intracellular
30 manipulation. Beneficially, the disclosed method is a simple and unique way to
selectively send desired cells and payloads to a predetermined location. Notably,
5
such method and system (namely, nanomotors) will be of interest to those working
on cell delivery, in-vivo theragnostic and regenerative medicine. For example, the
disclosed method uses specialized magnetic nanostructures to predispose cells to
undergo subsequent changes like bone formation at a target site.
5 It is to be appreciated that all the aforementioned implementation forms can be
combined. All steps which are performed by the various entities described in the
present application as well as the functionalities described to be performed by the
various entities are intended to mean that the respective entity is adapted to or
configured to perform the respective steps and functionalities. It will be appreciated
10 that features of the present disclosure are susceptible to being combined in various
combinations without departing from the scope of the present disclosure as defined
by the appended claims.
Additional aspects, advantages, features, and objects of the present disclosure
would be made apparent from the drawings and the detailed description of the
15 illustrative implementations construed in conjunction with the appended claims that
follow.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a flowchart of steps of a method of selectively manipulating cells using
magnetic nanostructures, in accordance with an embodiment of the present
20 disclosure;
FIG. 2A is a schematic illustration of an incubation process, in accordance with an
embodiment of the present disclosure;
FIG. 2B is an illustration of confocal microscopy images confirming internalization
of the magnetic nanostructure in the cells, in accordance with an embodiment of the
25 present disclosure.
FIGs. 3A-C are schematic illustrations of a remote activation process (or
mechanism of actuation) of a magnetic nanostructure, in accordance with an
embodiment of the present disclosure;
6
FIGs. 4A-E collectively illustrate manoeuvrability of the magnetic nanostructuresinternalized cells in different cell-substrate adhesion conditions, in accordance with
an embodiment of the present disclosure;
FIGs. 5A-E collectively illustrate manipulation of activated magnetic
5 nanostructures-internalized cells, in accordance with an embodiment of the present
disclosure;
FIGs. 6A-G collectively illustrate manipulation of a swarm of cells due to external
force and hydrodynamic interactions, in accordance with an embodiment of the
present disclosure; and
10 FIGs. 7A-C collectively illustrate an exemplary implementation of manipulation of
stem cell for bone regeneration, according to an embodiment of the present
disclosure, in accordance with an embodiment of the present disclosure.
The summary above, as well as the following detailed description of illustrative
embodiments, is better understood when read in conjunction with the appended
15 drawings. For the purpose of illustrating the present disclosure, exemplary
constructions of the disclosure are shown in the drawings. However, the present
disclosure is not limited to specific methods and instrumentalities disclosed herein.
Moreover, those skilled in the art will understand that the drawings are not to scale.
Wherever possible, like elements have been indicated by identical numbers.
20 Embodiments of the present disclosure will now be described, by way of example
only, with reference to the following diagrams wherein:
In the accompanying drawings, an underlined number is employed to represent an
item over which the underlined number is positioned or an item to which the
underlined number is adjacent. A non-underlined number relates to an item
25 identified by a line linking the non-underlined number to the item. When a number
is non-underlined and accompanied by an associated arrow, the non-underlined
number is used to identify a general item at which the arrow is pointing.
DETAILED DESCRIPTION OF EMBODIMENTS
7
The following detailed description illustrates embodiments of the present disclosure
and ways in which they can be implemented. Although some modes of carrying out
the present disclosure have been disclosed, those skilled in the art would recognize
that other embodiments for carrying out or practicing the present disclosure are also
5 possible.
FIG. 1 is a flowchart 100 of steps of a method of selectively manipulating cells
using magnetic nanostructures, in accordance with an embodiment of the present
disclosure. At step 102, live cells are incubated with the magnetic nanostructures to
obtain magnetic nanostructures-internalized cells. At step 104, the magnetic
10 nanostructures-internalized cells are remotely activated for cell manipulation by
applying an external force. The activated magnetic nanostructures-internalized cells
interact with surrounding non-active non-magnetic cells circulating in a fluid
medium to achieve active manipulation of a swarm of cells, and wherein the swarm
of cells comprises the activated magnetic nanostructures-internalized cells and the
15 non-active non-magnetic cells.
Herein, the phrase "selectively manipulating cells" refers to the precise and targeted
control of individual or groups of cells using magnetic nanostructures (or
nanomotors), within a controlled fluid medium. In an implementation, such precise
and targeted control (namely, manipulation) can involve a variety of actions,
20 including movement, positioning, or interaction with specific cellular components.
Selective manipulation is typically important in applications such as targeted drug
delivery to specific cells or tissues without affecting healthy ones, and so on.
FIG. 2A is a schematic illustration of an incubation process 200, in accordance with
an embodiment of the present disclosure. As shown, the live cells 202 (or cells) are
25 incubated with the magnetic nanostructures 204 in a fluid medium 206 to obtain
magnetic nanostructures-internalized cells 208. Herein, incubating live cells 202
with magnetic nanostructures 204 facilitates internalization of the magnetic
nanostructures 204 into the live cells 202. In this regard, the live cells are cultured
in a standard condition and then incubated with the magnetic nanostructures 204
30 (or functionalized magnetic nanostructures 204). During incubation process 200,
8
the magnetic nanostructures 204 (or functionalized magnetic nanostructures 204)
interact with the cell membranes of the live cells 202 to be internalized through
various cellular uptake mechanisms, such as endocytosis or phagocytosis. It may
be appreciated that the incubation process 200 is performed in sterile conditions to
5 prevent any potential infection to the live cells 202 and the magnetic nanostructures
204 or the magnetic nanostructures-internalized cells 208. Moreover, after the
incubation period, live cells 202 may be analysed to confirm the internalization of
the magnetic nanostructures 204 to obtain the magnetic nanostructures-internalized
cells 208, using conventional techniques such as microscopy, flow cytometry, or
10 magnetic resonance imaging (MRI).
FIG. 2B illustrates confocal microscopy images confirming internalization of the
magnetic nanostructure 204 in the cells 202. The optical section in the left panel
image is 6.2 µm above the bottom surface of the petri dish and 1.4 µm away from
the top of the cell, and the optical section in the right panel image is 2.1 µm above
15 the bottom surface of the petri dish and 5.5 µm away from the top of the cell. The
two confocal microscopy images confirm the internalization of nanomotors by cell
202 (herein HeLa cells) though the exact pathway of internalization.
In an implementation, the live cells 202 (or cells) may include stem cells, bones
cells, nerve cells, and so on. It may be appreciated that the live cells 202 (or cells)
20 are obtained from an individual or subject (such as a human or animal) by following
standard process conditions known to a person skilled in the art. Moreover,
obtaining and transferring processed cells, i.e., magnetic nanostructuresinternalized cells 208, back into the individual or subject are done in highly sterile
environment under the guidance of a trained professional.
25 The term "magnetic nanostructures" 204 as used herein refers to tiny (at nanoscale)
devices, namely, self-propelled swimmers, designed to move through fluid medium
206, such as biological fluids or cellular environments, under the influence of an
external force (such as magnetic field). In this regard, the magnetic nanostructures
204 exhibit superparamagnetism, that allows them to be easily magnetized in the
30 presence of an external magnetic field and lose their magnetization when the
9
magnetic field is removed. The magnetic nanostructures 204 are biocompatible and
cause no reactive oxidative stress or toxicity in the cells 202 in presence thereof.
Typically, the magnetic nanostructures 204 have dimension from few microns to
few hundreds of microns. Beneficially, the magnetic nanostructures 204 may be
5 designed to interact with living (or live) cells 202 to facilitate at least one of:
labelling cells allowing them to be manipulated under the influence of an external
magnetic field; separating target cells from a mixture, allowing for efficient cell
sorting; detecting specific biomarkers associated with diseases; targeted drug
delivery by guiding drug-loaded nanoparticles to specific cells or tissues;
10 therapeutic applications (such as hyperthermia treatments involving heating
targeted cells using magnetic fields). Additionally, beneficially, the magnetic
nanostructures may be isolated, after the predefined job of manipulation of cells
202, without any fluorescent tagging using the same cell manipulation technique
which is not trivial with existing techniques.
15 In an implementation, the magnetic nanostructures 204 are helical-shaped magnetic
nanomotors, wherein the helical-shaped magnetic nanomotors are magnetic
bioglass. The helical-shaped magnetic nanomotors are attributed with unique
propulsion capabilities for effectively navigating through complex biological
environments comprising biological fluids or tissues. In an implementation, the
20 helical-shaped magnetic nanomotors are composed of a glass-like material (namely,
bioglass) that incorporates magnetic elements (such as iron). Notably, the bioglass
is a bioactive material having ability to bond with biological tissues or cells.
Beneficially, the bioglass component enhances biocompatibility of the magnetic
nanostructures 204. Beneficially, due to biocompatibility or the bioglass component
25 and remotely controllability of the magnetic component of the helical-shaped
magnetic nanomotors, such magnetic nanostructures 204 may find application in
for example, measuring viscosity, elasticity, payload delivery, and so on.
In an implementation, the helical-shaped magnetic nanomotors have a length in a
range of 0.1-10.0 micrometre and a thickness in a range of 50-1000 nanometre. In
30 an implementation, the helical-shaped magnetic nanomotors have a length ranging
10
from 0.1, 0.5, 1, 1.5, 3, 6 or 9 up to 0.5, 1, 1.5, 3, 6, 9 or 10.0 micrometre and a
thickness ranging from 50, 100, 200, 400, 600 or 800 up to 100, 200, 400, 600, 800
or 1000 nanometre. In an example, the helical-shaped magnetic nanomotors has a
length of 3 micrometre and a thickness of 400 nanometre.
5 In an implementation, the method further comprises fabricating the magnetic
nanostructures 204 using silica, and depositing of a magnetic layer of iron on the
silica with a layer of adhesive material therebetween, wherein the adhesive material
is silver. Notably, the magnetic nanostructures 204 typically comprise a magnetic
layer or are composed of magnetic materials such as iron or alloys of iron, such as
10 iron oxide nanoparticles (e.g., magnetite or maghemite), and the like. Herein, base
material of the magnetic nanostructure 204 is silica (SiO2). Notably, silica is a
common material used in nanotechnology due to its stability, biocompatibility, and
ease of manipulation. Moreover, the magnetic layer of iron on the silica enables the
magnetic nanostructure 204 to respond well to external forces (such as magnetic
15 field). Furthermore, silver has excellent electrical conductivity and is often used in
nanotechnology applications. Herein, the layer of silver enhances the adhesion
between the silica and the magnetic layer of iron. Notably, the magnetic layer of
iron assigns a magnetic moment to the magnetic nanostructure 204 and when the
external force is applied, magnetic moment of the magnetic nanostructure 204
20 follows the external force which results in a motion of the magnetic nanostructure
204. In an implementation, the magnetic nanostructure 204 is fabricated using
GLancing Angle Deposition (GLAD) technique. It may be appreciated that the
magnetic nanostructure 204 may be fabricated using other techniques known to a
person skilled in the art. In an example, the magnetic nanostructure 204 is fabricated
25 using about 300-350 nm thick silica, over which a 5-10 nm thick layer of silver and
subsequently a 50 nm thick magnetic layer of iron are applied. Beneficially, silica,
silver and iron are biocompatible materials, thus magnetic nanostructure 204 can be
used for different kinds of biomanipulation.
In an implementation, additionally, the magnetic nanostructures 204 are surface
30 functionalized using biocompatible coatings, thereby allowing the surface
11
functionalized-magnetic nanostructures 204 to interact with biological entities, such
as cells 202. The term "biocompatible coatings" refers to materials that are nontoxic and do not induce adverse effects when in contact with biological entities,
such as cells 202. In an implementation, the biocompatible coatings include
5 polymers, proteins, peptides, ligands, other biomolecules, that promote cellular
uptake of the magnetic nanostructures 204. Beneficially, surface functionalization
(via chemical or physical methods) using biocompatible coatings enhances the
biocompatibility of the magnetic nanostructures 204, reduces potential toxicity, and
facilitates specific interactions with biological entities, such as cells 202. Moreover,
10 the use of ligands or biomolecules in the surface functionalization process enables
specific targeting of the magnetic nanostructures 204 to particular biological
entities, such as cells 202, thereby facilitating effective targeted drug delivery or
imaging.
In an implementation, the magnetic nanostructures 204 are designed as magnetic
15 nanoparticles, nanowires, nanodots, or other nano-sized magnetic materials. In an
implementation, the magnetic nanostructures 204 are designed as bi-helical
multistage nanomotors and implemented as vortices trap generators for non-contact
(remote) micro-manipulation of particles (such as cells 202) in a microfluidic
environment (such as fluid medium 206). The magnetic nanostructures 204, when
20 in-vivo, apply hydro-dynamic forces in the same order of magnitude as biological
matters, such as cells 202, when subjected to external forces. Beneficially, the
magnetic nanostructures 204 are capable of long-range transportation and
patterning precision of around 50 micron.
In an implementation, the method further comprises:
25 separating non-internalized and suspended magnetic nanostructures 204 from the
magnetic nanostructures-internalized cells 208 by using trypsinization and
centrifugation at 2000-2500 rpm for 10-15 minutes;
resuspending the magnetic nanostructures-internalized cells 208 in a fresh medium;
and
12
subjecting the resuspended magnetic nanostructures-internalized cells 208 to the
external force for the cell manipulation.
In this regard, separating non-internalized and suspended magnetic nanostructures
204 from the magnetic nanostructures-internalized cells 208 provides a population
5 of cells that have internalized the magnetic nanostructures 204. Trypsin is an
enzyme that breaks down proteins and detaches adherent cells 202 from a surface,
such as surface of culture dishes. Centrifugation followed by trypsinization
separates cells based on a density gradient thereof. Notably, the supernatant
containing non-internalized and suspended magnetic nanostructures 204 is
10 discarded and pellet at the bottom of centrifuge tube contains the magnetic
nanostructures-internalized cells 208. The magnetic nanostructures-internalized
cells 208 from pellet are resuspended in the fresh medium to obtain a homogeneous
cell suspension comprising only the magnetic nanostructures-internalized cells 208.
Subsequently, the magnetic nanostructures-internalized cells 208 are subjected to
15 external forces for selective manipulation thereof for various intended applications.
Beneficially, trypsinization, centrifugation, resuspension and manipulation allow
effective analysis of effects of the internalized magnetic nanostructures 204 on the
cells 202.
Herein, the term "remotely activating" refers to an ability to control, trigger, or
20 induce a specific response or behaviour in a biological system, such as cells 202,
from a distance, typically without direct physical contact. Such remote activation is
typically achieved through the application of external physical forces or stimuli that
interact with the properties of the internalized magnetic nanostructures 204 within
cells 202. Notably, the remote activation is provided from outside the biological
25 system, namely cells 202, to elicit a desired cellular response to facilitate targeted
drug delivery, cellular imaging and diagnostics, cell manipulation, biological
sensing. In an implementation, external devices, such as magnetic controllers or
systems, are used to generate the necessary external forces, such as magnetic fields,
for manipulating the magnetic nanostructures-internalized cells 208. Such external
13
devices may be designed to adaptively respond to the real-time position of the cells
202, allowing for dynamic adjustments to ensure precise targeting.
Herein, the term "external force" refers to a physical force applied from outside the
biological system, such as cells 202, to induce specific responses or movements
5 within the cells 202. Moreover, the external force is used for remote activation of
the magnetic nanostructures-internalized cells 208 without direct physical contact
therewith. Notably, the external force is controlled externally. Beneficially, the
remote activation techniques using external forces offer a non-invasive and precise
means to control cellular behaviour, making them valuable in various biomedical,
10 biotechnological, and research applications.
In an implementation, the external force applied on the magnetic nanostructuresinternalized cells 208 for the remote activation is one of: a magnetic field, an
electric field, an acoustic energy, or a combination thereof. The magnetic field is
used to remotely control and guide magnetic nanostructures 204 to specific
15 locations (within the body or in-vitro, during a research study) for applications like
targeted drug delivery, and so on. Typically, the magnetic nanostructures 204 align,
rotate, or move in response to the applied external magnetic field. Similarly, the
electric field is used to remotely activate and control magnetic nanostructures 204
having electrically-responsive properties. Typically, the application of the external
20 electric field induces changes in the charge distribution or orientation of
electrically-responsive components of the magnetic nanostructures 204, thereby
resulting in motion or structural changes in the magnetic nanostructures 204. The
acoustic energy (sound waves or ultrasound) creates pressure gradients which are
utilized to manipulate the magnetic nanostructures 204 by inducing motion or
25 changes in the properties of the magnetic nanostructures 204, allowing for remote
activation and effective targeting as ultrasound can penetrate tissues or cells 202.
Moreover, simultaneous use of magnetic field and acoustic energy may offer a
synergistic effect in providing enhanced control and precision. Alternatively, the
external force applied on the magnetic nanostructures-internalized cells 208 for the
30 remote activation is an optical (light) energy. Typically, the optical energy, such as
14
laser light, may be used to control the motion or behaviour of the magnetic
nanostructures 204 having photoactive components therein, through processes like
photothermal effects or photochemical reactions. Alternatively, the external force
applied on the magnetic nanostructures-internalized cells 208 for the remote
5 activation is a temperature change. Typically, changes in temperature, induced by
external means, may affect the behaviour of magnetic nanostructures 204 having
thermosensitive components therein, that undergo structural changes or exhibiting
altered mobility.
FIG. 3A-C are schematic illustrations of a remote activation process 300 (or
10 mechanism of actuation) of a magnetic nanostructure 204, in accordance with an
embodiment of the present disclosure. FIG. 3A illustrates an image of an activated
magnetic nanostructures 204 which is implemented as a helical-shaped magnetic
nanomotor under the effect of an external force, such as magnetic field. In FIG. 3A,
the arrows denoted by 'm' and 'B' depict the direction of movement of the magnetic
15 nanostructures 204 upon activation by an external force, such as magnetic field.
FIG. 3B illustrates a Scanning Electron Micrograph (SEM) image of the magnetic
nanostructures 204 which is implemented as a helical-shaped magnetic nanomotor.
FIG. 3C illustrates an energy dispersive spectroscopy (EDS) to confirm a magnetic
coating on the magnetic nanostructure 204. Scale bar = 1 µm.
20 In an implementation, the magnetic field is a rotating magnetic field, and the
rotating magnetic field is less than 500 Gauss. In an implementation, the rotating
magnetic field is in a range of 50-500 Gauss. In an implementation, the rotating
magnetic field is less than 80 Gauss. In an example, the rotating magnetic field is
80 Gauss. The rotating magnetic field refers to a magnetic field that varies in
25 intensity and direction as it moves through space in a circular or rotating motion.
The rotating magnetic field is used to induce controlled movements or rotations in
magnetic objects or systems, to achieve precise and dynamic motion without direct
physical contact. In magnetic actuation, rotating magnetic fields are typically
generated using electromagnetic coils or magnets. The arrangement of these coils
30 or magnets is designed to produce a magnetic field that rotates in a controlled
15
manner. In this regard, the rotating magnetic field is generated by energizing the
electromagnetic coils in a sequence with a 120-degree phase shift (three-phase
magnetic actuation). Moreover, the direction of rotation and the speed of the
rotating magnetic field may be controlled by adjusting the phase sequence and
5 frequency of applied current on the electromagnetic coils. Herein, the rotating
magnetic fields are often employed to actuate and manipulate magnetic
nanostructures-internalized cells 208 circulating in the fluid medium 206.
Beneficially, the magnetic nanostructures 204 when subjected to the rotating
magnetic field, can be directed to specific locations within the body for targeted
10 drug delivery. Moreover, the rotating magnetic field used for actuation is more
versatile and effective than gradient magnetic field or other magnetic fields.
Additionally, amplitude of magnetic field required for wireless powering of
magnetic nanostructures 204 are order of magnitude less for the rotating magnetic
fields than the gradient magnetic fields. Additionally, controllability and scaling up
15 is also simple and cost-effective for the rotating magnetic field.
Moreover, an actuation frequency of the external force, such as the magnetic field,
may vary depending on the viscosity of the fluid medium 206. An application of
the magnetic nanostructures 204 may therefore include functioning as a
microrheology probe. A microrheology probe typically is a tool or particle
20 employed in microrheology experiments to analyze the mechanical properties of
soft or complex materials, such as colloidal suspensions, polymer solutions, or
biological fluids, such as the fluid medium 206.
In an implementation, the remote activation of the magnetic nanostructuresinternalized cells 208 for cell manipulation comprises controlling manoeuvring of
25 the magnetic nanostructures-internalized cells 208 to a target site. Herein, the term
"controlling manoeuvring" refers to the ability to precisely guide, direct, or navigate
the movement of the magnetic nanostructures-internalized cells 208 by remotely
activating and manipulating the magnetic nanostructures 204 within them. The
external forces or stimuli influence the behaviour of the magnetic nanostructures
30 204 resulting in controlled movement of the magnetic nanostructures-internalized
16
cells 208. In this regard, the external forces, such as magnetic fields, guide or direct
the magnetic nanostructures-internalized cells 208 to the target site. Typically,
strength and orientation of the magnetic field may be adjusted multiple times to
influence the desired movement and directionality of the cells. Notably, the fluid
5 medium 206 also influence the behaviour of the magnetic nanostructures 204
resulting in controlled movement of the magnetic nanostructures-internalized cells
208.
FIG. 4A-E illustrate manoeuvrability of the magnetic nanostructures-internalized
cells in different cell-substrate adhesion conditions 402, 404 and 406, in accordance
10 with an embodiment of the present disclosure. Notably, cell-substrate adhesion
between the cell 202 and the substrate 408, such as the vessel wall or bottom surface
of the petri dish, also influence the manoeuvring of the magnetic nanostructuresinternalized cells 208 to a target site. In this regard, three different cell-substrate
adhesion conditions 400 may be considered to achieve totally distinct behaviours
15 in the manoeuvring of the magnetic nanostructures-internalized cells 208 to a target
site.
FIG. 4A depicts a first condition, i.e. adhered cells 402, where the magnetic
nanostructures-internalized cells 208 are entirely adhered to substrate 408. FIG. 4B
shows a well-controlled motion of the adhered cells 402 in the fluid medium 206.
20 The direction of applied magnetic field is also shown in the FIG. 4B.
Moreover, FIG. 4A depicts a second condition, i.e. semi-adhered cells 404, where
the magnetic nanostructures-internalized cells 208 are partially adhered to substrate
408. Herein, the semi-adhered cells 404 are obtained after cells are trypsinized after
24 hours of incubation. Notably, trypsin reduces/removes the adhesive forces that
25 anchor the cell 202 with the substrate 408, resulting in slightly attached or semiadhered cells 404 that are spherical in cross-section. As shown in FIG. 4C, the
manoeuvrability of magnetic nanostructures 204 inside semi-adhered cell 404 is
different than that in the adhered cells 402. Here, the magnetic nanostructures 204
sometimes tumble while propelling due to generation of small pockets in such semi30 adhered cells 404 where viscosity is less than rest of the cytosol. Hence, in the semi-
17
adhered cells 404, the magnetic nanostructures 204 move in combination of
tumbling and propulsion though the combination depends on the fluid medium 206
and may vary from cell to cell. As shown in FIG. 4D, due to the drag force
imbalance on the circular semi-adhered cell 404 while rotating around the x-axis,
5 the semi-adhered cell 404 rolls along the surface in y-direction. FIG. 4D depicts the
orientation of the magnetic nanostructures 204 that is different than that of the
applied direction which supports the tumbling configuration of the magnetic
nanostructures 204 inside the semi-adhered cell 404 while moving.
Moreover, FIG. 4A depicts a third condition, i.e. non-adhered cells 406, where the
10 magnetic nanostructures-internalized cells 208 are not adhered to substrate 408. As
shown in FIG. 4E, the magnetic nanostructures 204 can make the non-adhered cells
406 move in a controlled fashion using the magnetic field. Notably, in case of the
non-adhered cells 406, the interaction between magnetic nanostructures 204 and
non-adhered cells 406 is strong as there is no force to hold the non-adhered cells
15 406 in place, the entire non-adhered cells 406 starts to rotate when magnetic field
is applied (m ~ 10-14). As shown, non-adhered cells 406 which is actively moving
is shown within dashed circle and the inset shows the zoomed in image of the nonadhered cells 406 with the magnetic nanostructures 204 indicated by black arrow.
Herein, the direction of translational motion of the non-adhered cells 406 is parallel
20 to the plane of the applied magnetic field. The magnetic field is in xz plane as
indicated; however, the non-adhered cells 406 is moving along x axis whereas
magnetic nanostructures 204 translates perpendicular to the direction of the applied
field plane.
In an implementation, the controlled manoeuvrability of the magnetic
25 nanostructures-internalized cells 208 may be tracked and visualized using real-time
monitoring techniques, such as imaging technologies, to ensure accurate navigation
and targeting.
Moreover, the activated magnetic nanostructures-internalized cells 208 interact
with surrounding non-active non-magnetic cells 410 circulating in a fluid medium
30 206 to achieve active manipulation of a swarm of cells, wherein the swarm of cells
18
comprises the activated magnetic nanostructures-internalized cells 208 and the nonactive non-magnetic cells 410. Herein, the fluid medium 206 refers to the biological
environment of the subject. In an implementation, an in-vivo fluid medium 206
pertains to bodily fluids circulating within an organism, such as blood, serum,
5 plasma, lymph, cerebrospinal fluid, interstitial fluid blood, and so on; and an invitro fluid medium 206 pertain to artificial medium where the cells can interact.
Notably, the in-vitro fluid medium 206 is designed to mimic certain aspects of the
physiological environment found in-vivo, such as Dulbecco's Modified Eagle's
Medium (DMEM), a minimum essential medium (MEM), a RPMI 1640 medium,
10 Ham's F-12 nutrient mixture, a neural stem cell medium, an endothelial cell growth
medium, a complete cell culture medium, a physiological solution, and so on.
Herein, the activated magnetic nanostructures-internalized cells 208 refer to the
magnetic nanostructures-internalized cells 208 that have been remotely activated or
manipulated using external forces. The non-activated non-magnetic cells refer to
15 the cells that have not been internalized with the magnetic nanostructures 204, and
thus are not remotely activated or manipulated using external forces. In an
implementation, the non-active non-magnetic cells 410 may comprise cells semiadhered to magnetic nanostructure or cells completely lacking magnetic
nanostructure. Both the activated magnetic nanostructures-internalized cells 208
20 and the non-active non-magnetic cells 410 are present in the fluid medium 206,
where the activated magnetic nanostructures-internalized cells 208 actively interact
with the surrounding non-active non-magnetic cells 410 and guide or influence the
behaviour of the non-active non-magnetic cells 410, to achieve collective
movement or manipulation of the entire cell population, namely, the swarm of cells.
25 Beneficially, active manipulation of swarms of cells allows targeted drug delivery,
tissue engineering, exploration of emergent behaviours in cellular systems, realtime monitoring of the interactions within the swarm of cells.
FIGs. 5A-E illustrate manipulation of activated magnetic nanostructuresinternalized cells 208, in accordance with an embodiment of the present disclosure.
30 FIG. 5A shows manipulation of the activated magnetic nanostructures-internalized
cell 208 without affecting other cells, namely the non-active non-magnetic cells
19
410, in the vicinity. The trajectory of the activated magnetic nanostructuresinternalized cell 208 is indicated by solid lines. As shown, the activated magnetic
nanostructures-internalized cells 208 shows very precise manoeuvrability
depending upon the field applied, such that the activated magnetic nanostructures5 internalized cells 208 translates along downwards in y and then toward left in x,
upwards in y, right in x, respectively, and finally reach the same position from
which it started. The direction of translation and the plane of applied magnetic field
suggest that it is rolling kind of motion though the activated magnetic
nanostructures-internalized cells 208, in this case, do not touch the bottom surface
10 of the wall which is confirmed from the speed of the cells that is much less than the
speed it would be in case of proper rolling motion and which is ~ 50 um/s.
FIG. 5B illustrates that a rotating sphere, herein the activated magnetic
nanostructures-internalized cells 208, in bulk medium, herein the fluid medium 206,
does not produce translational motion due to its symmetry. However, the symmetry
15 is broken here due to the presence of surface, such as substrate 408, at the bottom.
Here, the sphere rotates in xz plane and there is a plane surface below it at a distance
h. Because of the proximity of the lower surface 208A of the sphere to the plane
surface, the lower surface 208A feels enhanced drag F_bottom, contrary to the
upper surface 208B which sees less drag F_top due to larger distance from the
20 surface. Effectively, the sphere, being a solid object, generates a resultant force
along x axis, F_d which lead the sphere to move along the same direction.
FIG. 5C depict frequency-dependence of speed of the activated magnetic
nanostructures-internalized cells 208. Notably, as the activated magnetic
nanostructures-internalized cells 208 is in low Reynolds number regime, linearity
25 of Stokes equation suggests that speed should also hold the same linearity with
frequency. The activated magnetic nanostructures-internalized cells 208 can rotate
to few Hz frequencies(<10Hz). As shown, a frequency dependent velocity is linear
up to certain frequency called a cut-off frequency. After the cut-off frequency, the
applied magnetic field and the magnetic moment of the magnetic nanostructures
30 204 start to phase slip and hence speed reduces thereafter. FIG. 5C presents data for
20
two different magnetic fields: 50 Gauss and 80 Gauss, and a cut-off frequency for
80 Gauss is larger than 50 Gauss as expected.
FIG. 5D depicts fluid flow pattern which is distinctly different than that of the
sphere rotating in bulk medium. This modified flow field induces a force on the
sphere which can be obtained by integrating stresses 𝜎௜௝ 5 , 𝑭 ൌ ∯ 𝝈. 𝒏 𝑑𝑠 , on the
surface of the sphere and the stress is calculated from the velocity information as
𝝈 ൌ െ𝑝𝕀 ൅ ሺ𝛁𝐮 ൅ ሺ𝛁𝐮ሻ்ሻ. This force should be equal to the drag force due to
translational motion of sphere moving with a speed v.
FIG. 5E illustrate determination of distance from lower surface of the activated
10 magnetic nanostructures-internalized cells 208 and the substrate 408. The colour
plot is obtained from finite element method while the white dots are the
experimental points related to velocities of activated magnetic nanostructuresinternalized cells 208 having different sizes. As shown, the velocity v of sphere (the
colour bar) is plotted against the distance from the lower surface of the sphere to
15 plane surface (d) along x axis and radius (r) along y. It confirms that the speed
increases as diameter of the sphere increases, and the distance of the wall reduces
in a non-trivial fashion. The velocities of activated magnetic nanostructuresinternalized cells 208 having different sizes are plotted on the same colour graph
(white circles) to identity the average distance between activated magnetic
20 nanostructures-internalized cells 208 and the substrate 408 of the wall. As shown,
the white circles spread from 1.2 µm to 1.4 µm and the average is close to 1.3 µm.
In an implementation, the remote activation of the magnetic nanostructuresinternalized cells 208 for cell manipulation comprises controlling manoeuvring of
the magnetic nanostructures-internalized cells 208 such that the non-active non25 magnetic cells 410 are guided to move using hydrodynamic interactions along a
path of motion of the magnetic nanostructures-internalized cells 208. Apart from
active manipulation of a swarm of cells as mentioned above, the non-active nonmagnetic cells 410 may be controllably manoeuvred via hydrodynamic interactions.
The hydrodynamic interactions refer to the complex interactions between fluid flow
30 and objects immersed in the fluid medium 206, such as the activated magnetic
21
nanostructures-internalized cells 208 and the non-active non-magnetic cells 410
present in the fluid medium 206. Typically, the hydrodynamic interactions arise due
to the movement of fluid medium 206 around objects, namely cells, immersed
therein. The viscosity of the fluid medium 206 influences the resistance to flow and
5 affects how objects interact with the surrounding fluid medium 206. The
hydrodynamic interactions include a drag force (i.e., resistance offered by the fluid
medium 206 to the object's motion), hydrodynamic forces (attractive and repulsive
components) between multiple objects in the fluid medium 206, particle-particle
interactions (resulting in clustering together or exhibiting cooperative behaviour),
10 cellular dynamics, sedimentation (settling in the fluid medium 206) and buoyancy
(ability to float in the fluid medium 206), and Brownian motion (resulting from
collisions and interactions between particles and the fluid medium 206 molecules).
As a result, as the activated magnetic nanostructures-internalized cells 208 move
along a specific path, the surrounding fluid medium 206 flow influences the
15 movement of the non-active non-magnetic cells 410, causing them to follow a
similar trajectory. In other words, the non-active non-magnetic cells 410 essentially
respond to the hydrodynamic cues generated by the activated magnetic
nanostructures-internalized cells 208. Beneficially, the collaborative motion of the
activated magnetic nanostructures-internalized cells 208 and the non-active non20 magnetic cells 410 are important for targeted interventions in biomedical and
biotechnological applications as mentioned above.
FIG. 6A-G illustrates manipulation of a swarm of cells due to external force and
hydrodynamic interactions, in accordance with an embodiment of the present
disclosure. FIG. 6A shows indirect manipulation of cells where the activated
25 magnetic nanostructures-internalized cells 208 is actively moving and when it
comes close to the non-active non-magnetic cells 410, it pushes them sideways and
as the activated magnetic nanostructures-internalized cells 208 goes away, it attracts
the non-active non-magnetic cells 410 towards its central path. Such, the non-active
non-magnetic cells 410 are manipulated by hydrodynamic interaction.
22
FIG. 6B depicts the fluid flow profile of the activated magnetic nanostructuresinternalized cells 208 as it rotates 602 and translates 604 at the same time, where
resultant fluid flow can be calculated by simple linear superposition 606 of the two
motions. In this regard,
5 FIGs. 6C and 6D depict fluid flow profile, along the plane through the centre of the
sphere and horizontal to the solid wall, for translational motion (near the surface)
and rotational motion, respectively. Here again we assumed cells to be solid sphere
near a wall with no slip boundary conditions at the interfaces. As shown, the spread
is more for rotational motion than the flow in translation motion of sphere.
10 However, the in-plane (xy plane) flow is low in the vicinity of the sphere for
rotational motion which is not the case for translational motion. Lastly, the flow
fields are high along the direction of motion for both the cases.
FIG. 6E shows the resultant flow field for rotational-translation motion of a sphere
near a wall where it can be clearly seen that flow field is extended around 75 µm
15 from the centre and hence cells, namely, the activated magnetic nanostructuresinternalized cells 208 and the non-active non-magnetic cells 410, which are within
this range, can feel the hydrodynamic force.
FIGs. 6F and 6G shows the simulated trajectory of a passively moving non-active
non-magnetic cells 410 and an experimental trajectory thereof, respectively, plotted
20 in the reference frame of actively moving activated magnetic nanostructuresinternalized cells 208. As shown, these two trajectories show excellent agreement.
In an implementation, the controlled manoeuvrability comprises active
manipulation of the swarm of cells, and wherein the controlled manoeuvrability
comprises actions selected from: approaching, pushing, carrying, dragging, or
25 releasing, or a combination thereof. Notably, the approaching, pushing, carrying,
dragging, or releasing, or a combination thereof actions may employ activated
magnetic nanostructures-internalized cells 208, the non-active non-magnetic cells
410, the fluid medium 206 or the target site. Herein, the action of approaching refers
to movement of the activated magnetic nanostructures-internalized cells 208
30 towards the non-active non-magnetic cells such as 410 or the target site as a result
23
of external forces and/or hydrodynamic interactions. The action of pushing may
refer to movement of the non-active non-magnetic cells 410 away from the
activated magnetic nanostructures-internalized cells 208 as a result of the
hydrodynamic interactions. Alternatively, the action of pushing may result in
5 movement of the non-active non-magnetic cells 410 away from the activated
magnetic nanostructures-internalized cells 208 in a desired direction. For example,
as the activated magnetic nanostructures-internalized cells 208 moves in a direction
because of applied external force (such as magnetic actuation), it induces the fluid
flow which can cause nearby/surrounding non-active non-magnetic cells 410 to
10 displace away from it.
The action of carrying refers to movement of the non-active non-magnetic cells 410
and/or the fluid medium 206 along with the activated magnetic nanostructuresinternalized cells 208 as a result of the hydrodynamic interactions or under the
influence of external forces on the activated magnetic nanostructures-internalized
15 cells 208, such as towards the target site. The action of dragging is similar to the
action of carrying, but along a surface, such as the vessel walls in the in-vivo
condition or a surface of a petri dish in the in-vitro setup, where the non-active nonmagnetic cells 410 and/or the fluid medium 206 move along with the activated
magnetic nanostructures-internalized cells 208 as a result of friction forces. The
20 action of releasing refers to removing a control over the non-active non-magnetic
cells 410 and/or the fluid medium 206 by the activated magnetic nanostructuresinternalized cells 208, or removing a control over the non-active non-magnetic cells
410 and/or the activated magnetic nanostructures-internalized cells 208 by the fluid
medium 206 to allow a free flow of the released object to achieve a desired outcome.
25 It may be appreciated that the controlled manoeuvrability employs various
combinations of the aforementioned actions in a coordinated sequence to achieve a
desired outcome. For example, the controlled manoeuvrability may employ
approaching and pushing action by the activated magnetic nanostructuresinternalized cells 208 on the non-active non-magnetic cells 410 to guide the swarm
30 of cells to the target site. Beneficially, selecting and combining these actions is of
importance in various applications such as targeted delivery, therapies, and so on.
24
In an implementation, the controlled manoeuvrability of the magnetic
nanostructures-internalized cells 208 and the non-active non-magnetic cells 410
comprises a translational motion and a rolling motion thereof. The translational
motion typically involves linear movement of an object, namely cells, from one
5 point to another without rotation. In other words, the cells move along a straight or
curved path without significant rotation. For example, cells undergoing
translational motion move in a direction guided by the external forces, in response
to gradients, stimuli, or other external factors. The rolling motion typically is a type
of rotational movement where an object, namely cells, rotates around its axis while
10 simultaneously moving linearly along a surface or substrate (such as vessel walls
(in-vivo) or bottom surface of a petri dish (in-vitro)). Notably, the magnetic layer
assigns a magnetic moment to the magnetic nanostructures-internalized cells 208
and when the rotating magnetic field is applied, the magnetic moment of the helicalshaped magnetic nanomotor follows the rotating magnetic field which results in the
15 rotational motion which, in turn, leads to a translational motion due to translationrotational coupling of the helical-shaped magnetic nanomotor internalized by the
cell. For instance, the magnetic nanostructures-internalized cells 208 may be guided
by a rotating magnetic field, inducing translational and rolling motions as they
move along a predefined path. The non-active non-magnetic cells 410, influenced
20 by hydrodynamic interactions with the actively moving cells, may exhibit similar
translational and rolling motions, leading to coordinated movement of the swarm
of cells within a system. Beneficially, both translational and rolling motions in
controlled manoeuvrability offers a more dynamic and versatile means of directing
the movement of cells with precision in various environments, contributing to, for
25 example, facilitating targeted drug delivery and other applications in fields like
nanomedicine and cellular therapies.
In an implementation, the translational motion of the magnetic nanostructuresinternalized cells 208 and the non-active non-magnetic cells 410 is parallel to a
plane of the applied field corresponding to the external force, and wherein the
30 parallel translational motion is caused from an increased first drag experienced by
a lower surface of the magnetic nanostructures-internalized cells 208 as compared
25
to a second drag experienced by a top surface of the magnetic nanostructuresinternalized cells 208. Notably, the external force, namely the magnetic field,
influences the movement of the swarm of cells in a specific direction parallel to the
plane defined by the applied external force. In other words, the swarm of cells move
5 in a direction aligned with the orientation of the magnetic field. Moreover, the
swarm of cells move in the parallel translational motion as influenced by a
difference in drag forces experienced by the lower surface compared to the top
surface of these cells. Typically, the drag forces act on the cells moving through the
fluid medium 206. Notably, the lower surface of the magnetic nanostructures10 internalized cells 208 experiences an increased first drag compared to the second
drag experienced by the top surface thereof. Moreover, such differential drag force
may be due to factors such as the orientation or shape of the cells, characteristics of
the fluid medium 206, or other factors. As a result of the differential drag forces,
the magnetic nanostructures-internalized cells 208 undergo a translational motion
15 in the direction aligned with the orientation of the increased drag forces and the
applied external forces.
In an implementation, a speed of the translational motion of the magnetic
nanostructures-internalized cells 208 and the non-active non-magnetic cells 410 is
in a range of 1-60 µm/s. In an implementation, the speed of the translational motion
20 of the magnetic nanostructures-internalized cells 208 and the non-active nonmagnetic cells 410 is in a range of 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or 55 µm/s
up to 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 µm/s. In an implementation, the
speeds of the translational motion of the magnetic nanostructures-internalized cells
208 and the non-active non-magnetic cells 410 may be same or different. In an
25 example, the speed of the translational motion of the magnetic nanostructuresinternalized cells 208 is 40-60 µm/s and the non-active non-magnetic cells 410 is
40 µm/s.
In an implementation, the speed increases with an increase in diameter of the
magnetic nanostructures-internalized and a decrease in distance between the
30 magnetic nanostructures-internalized cells 208 and a vessel wall of the fluid
26
medium 206. Larger cells generally experience higher drag forces when moving
through the fluid medium 206, resulting in a slower speed thereof. However, with
changes in the large cell's shape or surface properties that reduce drag, the speed of
cell may be increased. Closer proximity to the vessel wall or the bottom surface of
5 the petri dish may result in the Magnus effect, potentially increasing the speed of
the cells. Thus, there may be an optimal combination of cell diameter and distance
to the vessel wall that maximizes speed.
In an implementation, the method further comprises coating the magnetic
nanostructures-internalized cells 208 with a biomaterial selected from any of:
10 calcium silicate, hydroxyapatite, polylactic acid, polycaprolactone, gelatin,
chitosan, decellularized extracellular matrix, graphene and graphene oxide, or any
suitable combination thereof. Beneficially, the aforementioned biomaterials
improve biocompatibility, enhance stability, and enable specific functionalities,
such as bioavailability, degradation characteristics, mechanical properties, effective
15 integration of the biomaterial with the magnetic nanostructures-internalized cells
208, increasing speed, of the magnetic nanostructures-internalized cells 208. The
biomaterials you've listed offer diverse properties and applications. In an example,
calcium silicate and hydroxyapatite may be used in bone tissue engineering or bone
and dental implant coatings due to their similarity to the mineral component of
20 natural bone, biocompatibility, and ability to promote osteogenesis. In another
example, polylactic acid (PLA) and Polycaprolactone (PCL) are biodegradable
polymers often used in medical implants, drug delivery systems, and tissue
engineering. In yet another example, gelatin (derived from collagen) and chitosan
(derived from chitin) are used in drug delivery, wound healing, and tissue
25 engineering due to their biocompatibility. In still another example, decellularized
extracellular matrix (ECM) provides a biomimetic environment for cells, promoting
cell adhesion, growth, and differentiation. In still another example, graphene and
graphene oxide is known for their unique mechanical, electrical, and biocompatible
properties, and thus are often used in biosensors, drug delivery, and tissue
30 engineering. Moreover, a combination of two or more of such biomaterials may be
selected depending on the specific application, the desired properties, and the
27
intended function of the coated cells. For instance, in regenerative medicine or
tissue engineering, coatings like hydroxyapatite, calcium silicate, or decellularized
ECM may enhance the interaction of cells with the surrounding environment. In
drug delivery, polymers like polylactic acid (PLA) and polycaprolactone (PCL)
5 may provide controlled release properties.
In summary, the present disclosure focuses to obtain different applications from
helical-shaped magnetic nanomotors (or nanostructures). In this regard, cellsubstrate interaction is altered, and then same rotating magnetic field is applied to
actuate the nanomotors. Apart from the active manipulation, it is also possible to
10 manipulate cells which do not contain nanomotor via pure hydrodynamic
interaction. Notably, manoeuvrability of nanomotors inside the living cells semiadhered to the substrate shows tumbling motion in some regions of a semi-adhered
cell where the cytoskeleton dynamics is different than the rest of the cell. When
cell-substrate interaction is reduced to nil, a totally new phenomenon of cell
15 manipulation is observed. As the bottom wall is not supporting the cell, when a
rotating magnetic field is applied to the cell-substrate (system), generated torque
makes the cell to rotate along with the applied rotating magnetic field and being
very close to the wall (~1.3 µm), the rotating cell feels a force due to hydrodynamic
interaction with the bottom wall which leads to the translational motion as it rotates.
20 The activated nanomotor bearing cell rotates and translates near a wall and
influence the nearby/surrounding cells, such as non-active cells bearing no
magnetic nanostructure, due to fluid structure interaction, thereby manipulating a
swarm of cells passively using hydrodynamics interaction between actively moving
cell and the cells that do not contain nanomotor. Hence, the disclosed method is
25 able to fulfil all aspects of a novel cell manipulation technique which does not
require fluorescent tagging of cells and require very low magnetic field which is
benign to living cells 202.
FIG. 7A-C illustrate an exemplary implementation of manipulation of stem cell for
bone regeneration in mice teeth 700, according to an embodiment of the present
30 disclosure. In this regard, biohybrid stem cells are produced, by incubating the stem
28
cells with magnetic nanostructures, namely, magnetic nanostructure-internalized
cells 208 that can be magnetically manoeuvred due to the presence of magnetic
nanostructures inside the stem cells. The magnetic nanostructures also referred to
as CALBOTS are coated (surface modification) with calcium silicate to trigger
5 bone growth, such as to mitigate issue of tooth fracture and tooth regeneration. The
dental pulp is one of the few areas where stem cells are still found in human adults.
FIG. 7A shows accumulation of stem cells, as a result of magnetic actuation, in a
region of fracture 702 to quicken bone growth in the mice teeth.
FIG. 7B depicts a single magnetic nanostructures-internalized cells 208, propelling
10 a plurality of non-magnetic cells 410 towards the fracture site of the mice teeth 700.
The method of using hydrodynamic interactions to manoeuvre the non-magnetic
cells 410 can be used synergistically to amplify the cell delivery method.
FIG. 7C illustrates a 24-well plate as a proof of predisposition of stem cells and 410
to bone formation due to presence of specialized magnetic nanostructures.
15 Predisposition to bone formation due to presence of calcium silicates in the
magnetic biostructures. The controlled bone growth can be seen in the 4 dots in the
‘Cells+calbot+mag’ column of the array where the dots correspond to the place
where the calbots were concentrated using magnetic field.
Modifications to embodiments of the present disclosure described in the foregoing
20 are possible without departing from the scope of the present disclosure as defined
by the accompanying claims. Expressions such as "including", "comprising",
"incorporating", "have", "is" used to describe and claim the present disclosure are
intended to be construed in a non-exclusive manner, namely allowing for items,
components or elements not explicitly described also to be present. Reference to
25 the singular is also to be construed to relate to the plural. The word "exemplary" is
used herein to mean "serving as an example, instance or illustration". Any
embodiment described as “exemplary” is not necessarily to be construed as
preferred or advantageous over other embodiments and/or to exclude the
incorporation of features from other embodiments. The word "In an
30 implementation" is used herein to mean "is provided in some embodiments and not
29
provided in other embodiments". It is appreciated that certain features of the present
disclosure, which are, for clarity, described in the context of separate embodiments,
may also be provided in combination in a single embodiment. Conversely, various
features of the present disclosure, which are, for brevity, described in the context of
5 a single embodiment, may also be provided separately or in any suitable
combination or as suitable in any other described embodiment of the disclosure.
EXPERIMENTAL PART
Fabrication of nanomotors: The helical-shaped magnetic nanomotors
10 (nanostructures) were fabricated using Glancing Angle Deposition (GLAD)
technique. It is a versatile vapour deposition technique to make 3D porous
nanostructured films, from where helical-shaped magnetic nanomotors could be
obtained. The width of the helical-shaped magnetic nanomotors depended on the
seeding used for pre-patterning the substrate before the GLAD growth. For two
15 types of helical-shaped magnetic nanomotors that were used, seeding layers of two
different dimensions were used. For the bigger one, a monolayer of polystyrene
(PS) bead (from Spherotech) of 700 nm diameter was obtained. A Langmuir–
Blodgett system (Apex Instruments, Kolkata) was used to deposit the PS beads on
a piranha cleaned silicon wafer. Thereafter, the substrate was placed in a RF plasma
20 chamber and etched for 12 min at 10.5 W. This step was necessary to increase the
separation between the PS beads which is a crucial parameter in nanomotor
fabrication, and to reduce the diameter of the PS beads to around 500 nm. This
substrate was used in GLAD for further processes. Silica helices were grown on
these substrates using GLAD where a material flux comes at an extreme angle (~5
25 degrees) while the substrate rotated at a certain rate depending on the geometry of
helices. Feature size of the seeding layer determined its thickness, while pitch of
these helices was determined by the rotation rate of substrate. The third step of
fabrication was to lay these nanomotors down on a freshly cleaned silicon wafer
and deposit ~ 50 to 70 nm magnetic layer (herein, iron) using thermal treatment.
30 These magnetic material-coated nanomotors were sonicated out in a solution.
30
Cell culture: HeLa (ATCC) were cultured in DMEM supplemented with 10% FBS,
at 37ºC with 5% CO2. 0.075 million cells were grown overnight in 35 mm glass
bottom dishes. 106 nanomotors were incubated with the cells for 24 hours in culture
medium. After 24 hours, the cells were washed thrice with PBS to remove the non5 internalized helix-shaped magnetic nanomotors. The dish was then placed inside a
Helmholtz coil, placed in an optical microscope and a rotating magnetic field was
applied.
Internalization study: HeLa cells (0.1 million) were grown overnight in a glass
bottom dish. Cells were serum starved for 30 mins prior to transfection using serum
10 free media. A transfection mix was prepared, consisting of 2µg GFP-βactin DNA,
4µL of Turbofect Transfection Reagent (Thermo Fischer) and 100 µL of PBS and
added to the cells. After 6 hours, serum free medium was replaced with DMEM
supplemented with 10% FBS and after 24 hours, 106 helix-shaped magnetic
nanomotors were added to the cells, for another 24 hours. After which, the cells
15 were washed with PBS to remove the non-internalized helix-shaped magnetic
nanomotors. Notably, the magnetic nanomotors may have entered the cells via
phagocytosis, or a similar mechanism. Confocal imaging was performed on Zeiss
LSM 880 confocal microscope. A Z-stack of the optical sections was acquired with
100 nm z resolution in the GFP channel for actin as well as in bright field to
20 visualizing the helix-shaped magnetic nanomotor. The helix-shaped magnetic
nanomotor was pseudo coloured in the Z stack and a 3D rendering of the stack was
created with ImageJ/ Fiji.
Cell-substrate interaction modification: 0.075 million HeLa cells or Bovine Aorta
Endothelial Cells (BAEC) were grown overnight in 35 mm glass bottom dishes.
25 106 helix-shaped magnetic nanomotors were incubated with the cells for 24 hours.
After 24 hours, the cells were washed with PBS, to remove the non-internalized
helix-shaped magnetic nanomotors. For experiments with adhered cells, the same
dish was used. For other two types (i.e. semi-adhered and non-adhered), cells were
then detached by trypsinization and centrifuged at 2000 rpm for 10 mins and were
30 resuspended in fresh medium. Trypsin is a member of serine protease family, and
31
it cleaves peptides on the C-terminal side of either lysine or arginine amino acid
with the help of calcium ion present in the medium and thus reduces / removes the
adhesive forces that anchor the cell with the substrate. The cells were then seeded
on a 35 mm glass bottom dish and the experiment was performed after 2 hours for
5 semi-adhered cell and immediately after trypsinzing for non-adhered cells. As the
interaction between helix-shaped magnetic nanomotors and cell is strong and now
there is no force to hold the cell in place (after trypsinization), the entire cell starts
to move (namely, rotate) when external force (rotating magnetic field) is applied.
Method of actuation: Once helix-shaped magnetic nanomotors were internalised by
10 the cells, they were actuated using a rotating magnetic field generated by a triaxial
Helmholtz coil. This coil could generate rotating field in arbitrary direction, though,
in these set of experiments, field which was rotating in a plane perpendicular to the
focusing plane of the microscope was used. The actuation principle of nanomotors
is based on the intrinsic rotation-translation coupling of a helical structure at low
15 Reynolds numbers. Applied rotating magnetic field B, generates a magnetic torque,
τ = m × B, on the nanomotor (where m is the magnetic moment of the helix). As
the interaction between helix-shaped magnetic nanomotors and cell is strong and
now there is no force to hold the cell in place (after trypsinization), the entire cell
starts to rotate when rotating field is applied. As a result, nanomotor starts to rotate
20 and due to its rotation-translation coupling, it moves forward in a direction
perpendicular to plane of rotating field and hence always remained in the imaging
plane of the microscope.
In an example, the above-described method was used to actively manipulate stem
cells. Notably, bone loss/fracture is a common problem in geriatric patients.
25 Targeted delivery of stem cells can trigger bone regeneration. In this regard,
biohybrid stem cells were produced that were magnetically manoeuvred due to the
presence of magnetic nanostructures inside the stem cells. Such nanostructures
(called CALBOTs) were coated with calcium silicate to trigger bone growth, such
as in case of tooth fracture and tooth regeneration. Although teeth are nonessential
30 for life and thus not considered a significant target for regenerative medicine
32
research, however, teeth are ideal for testing new cell-based treatments. The dental
pulp is one of the few areas where stem cells are still found in human adults. The
disclosed method was used to accumulate stem cells in a region of fracture to
quicken bone growth. Hydrodynamic interactions were used to manoeuvre non5 magnetic cells along with external force activation of the magnetic nanostructureinternalized stem cells to synergistically amplify the cell delivery method. The
single magnetic cell was used to bring a plurality of cells near the fracture site.
33
CLAIMS
We claim:
1. A method of selectively manipulating cells using magnetic nanostructures 204,
the method comprising:
5 incubating live cells 202 with the magnetic nanostructures to obtain magnetic
nanostructures-internalized cells 208; and
remotely activating the magnetic nanostructures-internalized cells for cell
manipulation by applying an external force,
wherein the activated magnetic nanostructures-internalized cells interact with
10 surrounding non-active non-magnetic cells 410 circulating in a fluid medium 206
to achieve active manipulation of a swarm of cells,
and wherein the swarm of cells comprises the activated magnetic nanostructuresinternalized cells and the non-active non-magnetic cells.
2. The method as claimed in claim 1, wherein the external force applied on the
15 magnetic nanostructures-internalized cells 208 for the remote activation is one of:
a magnetic field, an electric field, an acoustic energy, or a combination thereof.
3. The method as claimed in claim 1, wherein the remote activation of the magnetic
nanostructures-internalized cells 208 for cell manipulation comprises controlling
manoeuvring of the magnetic nanostructures-internalized cells to a target site.
20 4. The method as claimed in claim 1, wherein the remote activation of the magnetic
nanostructures-internalized cells 208 for cell manipulation comprises controlling
manoeuvring of the magnetic nanostructures-internalized cells such that the nonactive non-magnetic cells 410 are guided to move using hydrodynamic interactions
along a path of motion of the magnetic nanostructures-internalized cells.
25 5. The method as claimed in claim 4, wherein the controlled manoeuvrability
comprises active manipulation of the swarm of cells, and wherein the controlled
manoeuvrability comprises actions selected from: approaching, pushing, carrying,
dragging, or releasing, or a combination thereof.
34
6. The method as claimed in claim 5, wherein the controlled manoeuvrability of the
magnetic nanostructures-internalized cells 208 and the non-active non-magnetic
cells 410 comprises a translational motion and a rolling motion thereof.
7. The method as claimed in claim 6, wherein the translational motion of the
5 magnetic nanostructures-internalized cells 208 and the non-active non-magnetic
cells 410 is parallel to a plane of the applied field corresponding to the external
force, and wherein the parallel translational motion is caused from an increased first
drag experienced by a lower surface of the magnetic nanostructures-internalized
cells as compared to a second drag experienced by a top surface of the magnetic
10 nanostructures-internalized cells.
8. The method as claimed in claim 6, wherein a speed of the translational motion of
the magnetic nanostructures-internalized cells 208 and the non-active non-magnetic
cells 410 is in a range of 1-60 µm/s.
9. The method as claimed in claim 1, further comprising:
15 separating non-internalized and suspended magnetic nanostructures 204 from the
magnetic nanostructures-internalized cells 208 by using trypsinization and
centrifugation at 2000-2500 rpm for 10-15 minutes;
resuspending the magnetic nanostructures-internalized cells in a fresh medium; and
subjecting the resuspended magnetic nanostructures-internalized cells to the
20 external force for the cell manipulation.
10. The method as claimed in claim 2, further comprising coating the magnetic
nanostructures-internalized cells 208 with a biomaterial selected from any of:
calcium silicate, hydroxyapatite, polylactic acid, polycaprolactone, gelatin,
chitosan, decellularized extracellular matrix, graphene and graphene oxide, or any
25 suitable combination thereof.
11. The method as claimed in claim 1, wherein the magnetic nanostructures 204 are
helical-shaped magnetic nanomotors, wherein the helical-shaped magnetic
nanomotors are magnetic bioglass.
35
12. The method as claimed in claim 11, further comprising fabricating the magnetic
nanostructures 204 using silica, and depositing of a magnetic layer of iron on the
silica with a layer of adhesive material therebetween, wherein the adhesive material
is silver.
5 13. The method as claimed in claim 11, wherein the helical-shaped magnetic
nanomotors have a length in a range of 0.1-10.0 micrometre and a thickness in a
range of 50-1000 nanometre.
14. The method as claimed in claim 2, wherein the magnetic field is a rotating
magnetic field, and the rotating magnetic field is less than 500 Gauss.
10 15. The method as claimed in claim 8, wherein the speed increases with an increase
in diameter of the magnetic nanostructures-internalized 208 and a decrease in
distance between the magnetic nanostructures-internalized cells and a vessel wall
of the fluid medium 206.