U.S. patent application number 15/579084 was filed with the patent office on 2018-06-21 for mechano-sensitive microcapsules for drug delivery.
This patent application is currently assigned to The Trustees Of The University Of Pennsylvania. The applicant listed for this patent is The Trustees Of The University Of Pennsylvania. Invention is credited to George R. Dodge, Daeyeon Lee, Robert L. Mauck, Bhavana Mohanraj, Fuquan Tu.
Application Number | 20180169024 15/579084 |
Document ID | / |
Family ID | 57441725 |
Filed Date | 2018-06-21 |
United States Patent
Application |
20180169024 |
Kind Code |
A1 |
Lee; Daeyeon ; et
al. |
June 21, 2018 |
MECHANO-SENSITIVE MICROCAPSULES FOR DRUG DELIVERY
Abstract
Embodiments of the present invention relate to
mechanically-activated microcapsules (MAMCs) for controlled
drug-delivery, wherein the MAMCs release one or more active
ingredients in response to mechanical stimuli in a subject's body.
The MAMCs provide a platform for stimulating biological
regeneration, biological repair, modifying disease, and/or
controlling disease in mechanically-loaded musculoskeletal
tissues.
Inventors: |
Lee; Daeyeon; (Wynnewood,
PA) ; Mauck; Robert L.; (Philadelphia, PA) ;
Dodge; George R.; (Philadelphia, PA) ; Tu;
Fuquan; (Philadelphia, PA) ; Mohanraj; Bhavana;
(Philadelphia, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Trustees Of The University Of Pennsylvania |
Philadelphia |
PA |
US |
|
|
Assignee: |
The Trustees Of The University Of
Pennsylvania
Philadelphia
PA
|
Family ID: |
57441725 |
Appl. No.: |
15/579084 |
Filed: |
June 1, 2016 |
PCT Filed: |
June 1, 2016 |
PCT NO: |
PCT/US2016/035220 |
371 Date: |
December 1, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62169286 |
Jun 1, 2015 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 9/1075 20130101;
A61K 9/5031 20130101; A61K 9/5089 20130101; A61K 9/0024 20130101;
A61K 38/1841 20130101; B01J 13/02 20130101; A61P 19/04
20180101 |
International
Class: |
A61K 9/50 20060101
A61K009/50; A61K 38/18 20060101 A61K038/18; A61P 19/04 20060101
A61P019/04 |
Claims
1. A method of tuning the rupture profiles of
mechanically-activated microcapsules to deliver a therapy to a
subject comprising: identifying one or more rupture profiles that
will provide a therapy to a subject by enabling the
mechanically-activated microcapsules to rupture in response to one
or more mechanical loads that are expected to be applied to the
mechanically-activated microcapsules after they have been
administered to the subject, and creating mechanically-activated
microcapsules that have said one or more rupture profiles, wherein
each mechanically-activated microcapsule comprises one or more
active ingredients encapsulated inside a shell, wherein said
mechanically-activated microcapsules are designed to rupture and
release a therapeutically effective amount of said one or more
active ingredients when the one or more mechanical loads are
applied to said mechanically-activated microcapsules.
2. The method according to claim 1, wherein creating the
mechanically-activated microcapsules comprises providing shell
thicknesses that enable the mechanically-activated microcapsules to
rupture and release the therapeutically effective amount of said
one or more active ingredients when the one or more mechanical
loads are applied to said mechanically-activated microcapsules.
3. The method according to claim 1, wherein creating the
mechanically-activated microcapsules comprises adding one or more
plasticizers to the shell.
4. The method according to claim 1 further comprising embedding the
mechanically-activated microcapsules within a matrix material.
5. The method according to claim 1, wherein one or more of the
administered mechanically-activated microcapsules are designed to
rupture and release the therapeutically effective amount of said
one or more active ingredients after a plurality of mechanical
loads is applied over time.
6. The method according to claim 5, wherein the plurality of
mechanical loads applied over time is at least one of a regimen of
different mechanical loads applied over time, or a regimen of
identical loads repeated over time.
7. A method of using mechanically-activated microcapsules for drug
delivery comprising: delivering the mechanically-activated
microcapsules (MAMCs) to a region of a subject's body, wherein the
mechanically-activated microcapsules have one or more rupture
profiles that enable the mechanically-activated microcapsules to
rupture in response to one or more mechanical loads in said region
of the subject's body.
8. The method according to claim 7, wherein delivering the MAMCs to
said region of the subject's body comprises injecting the MAMCs
into said region.
9. The method according to claim 7 comprising delivering the MAMCs
to a joint.
10. The method according to claim 7 comprising delivering the MAMCs
to a knee joint, elbow joint, shoulder joint, wrist joint, ankle
joint or hip joint.
11. The method according to claim 7 comprising delivering the MAMCs
to non-joint tissue.
12. The method according to claim 7, wherein the MAMCs are embedded
within a matrix material, and wherein delivering the MAMCs to said
region of the subject's body comprises implanting the matrix
material into said region.
13. The method according to claim 7, wherein the MAMCs have a
plurality of different rupture profiles.
14. The method according to claim 7, wherein the one or more
rupture profiles of the MAMCs enable the MAMCs to rupture after
exposure to a single mechanical load.
15. The method according to claim 7, wherein the one or more
rupture profiles of the MAMCs enable the MAMCs to rupture after
each of multiple exposures to mechanical loads.
16. The method according to claim 7, wherein the MAMCs comprise one
or more active therapeutics , wherein the active therapeutics
comprise anti-catabolic compounds, anabolic compounds,
anti-inflammatory compounds, chondrogenic factors, and growth
factors.
17. The method according to claim 16, wherein the active
therapeutics comprise at least one of transforming growth factors,
fibroblast growth factors, connective tissue growth factors,
insulin-like growth factors, or bone morphogenetic proteins.
18. The method according to claim 7, wherein the MAMCs comprise
polymeric shells comprising poly(lactic-co-glycolic)acid
(PLGA).
19. The method according to claim 11, wherein the MAMCs delivered
to non-joint tissue comprise one or more active ingredients for
wound healing.
20. The method according to claim 16, wherein the active
therapeutics are designed to control or modify a disease in
mechanically-loaded musculoskeletal tissues.
21. A composition comprising a mixture of mechanically-activated
microcapsules having a plurality of rupture profiles.
22. The composition according to claim 21, wherein the
mechanically-activated microcapsules have more than one shell
thickness and/or more than one shell composition.
23. The composition according to claim 21, wherein the composition
further comprises a pharmaceutically acceptable carrier.
24. The composition according to claim 21, wherein the composition
further comprises one or more excipients.
25. The composition according to claim 21, wherein the composition
is embedded within a matrix material.
26. A method of making the composition of claim 21 comprising
mixing the mechanically-activated microcapsules with a
pharmaceutical carrier and one or more optional excipients.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application No. 62/169286, entitled MECHANO-SENSITIVE MICROCAPSULES
FOR DRUG DELIVERY, filed Jun. 1, 2015, the contents of which are
incorporated by reference herein in their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to mechano-sensitive
microcapsules for drug delivery, and methods of using the same.
BACKGROUND OF THE INVENTION
[0003] In comparison to conventional systemic delivery of
therapeutics, controlled drug delivery has several advantages,
including localized delivery to specific locations, maintenance of
drug concentrations within a desired therapeutic range, and
preservation of therapeutic activity for long-term administration.
One particularly desirable feature of these systems is
self-regulation, wherein physiological feedback actively controls
release kinetics. Self-regulating delivery systems often rely on
internal triggers for release, such as temperature, pH-sensitivity,
enzyme-substrate reactions, or chemical (hydrolysis) reactions.
Recent advances in microencapsulation-based ("core-shell") drug
delivery systems have used these stimuli-responsive approaches; for
example, lipid shells melted at 37.degree. C. have been used to
release anti-cancer drugs and shell pH sensitivity has directed
antibiotic release in the gastrointestinal tract.
[0004] However, there are no controlled delivery vehicles that have
been tuned for triggered release in response to mechanical loading,
deformation, or stress. Tissues within the body experience
mechanical perturbation across multiple force magnitudes and length
scales, from mechanotransduction at the cellular level to the
dynamics of whole joints. These forces in most tissues are
responsible for maintaining tissue integrity, and can initiate
degenerative processes at supra-physiologic levels. Thus, there
remains a need for drug delivery systems that can be activated in
response to mechanical loading, whether it is incurred during
rehabilitation, normal activities of daily living, or under
conditions likely to produce tissue damage.
SUMMARY OF THE INVENTION
[0005] An embodiment of the present invention relates to a method
of tuning the rupture profiles of mechanically-activated
microcapsules MAMCs to deliver a therapy to a subject. The method
includes the steps of identifying one or more rupture profiles that
will provide a therapy to a subject based on mechanical
loads/stresses/strains (and/or based on the number of loading
events/timing of loadings) that are expected to be applied to
mechanically-activated microcapsules after they have been
administered to the subject, and
creating mechanically-activated microcapsules that have said one or
more rupture profiles. Each mechanically-activated microcapsule
comprises one or more active ingredients encapsulated inside a
shell, and the mechanically-activated microcapsules are designed to
rupture and release a therapeutically effective amount of said one
or more active ingredients when said mechanical
loads/stresses/strains are applied to said mechanically-activated
microcapsules.
[0006] Another embodiment of the present invention relates to a
method of using mechanically-activated microcapsules for drug
delivery. The method includes delivering the mechanically-activated
microcapsules (MAMCs) to a region of a subject's body. The
mechanically-activated microcapsules have one or more rupture
profiles that enable the mechanically-activated microcapsules to
rupture in response to one or more mechanical loads in said region
of the subject's body.
Another embodiment of the present invention relates to a
composition including a mixture of mechanically-activated
microcapsules having a plurality of rupture profiles, e.g., for the
purpose of delivering multiple agents suitable for multiple
functions and temporal control of their cumulative release.
BRIEF DESCRIPTION OF THE DRAWINGS
[0007] FIG. 1a provides a schematic illustration of an embodiment
of a water/oil/water (W/O/W) double emulsion generation from a
capillary microfluidic device according to aspects of the present
invention.
[0008] FIG. 1b provides a microscopy image of an embodiment of
water/oil/water (W/O/W) double emulsion generation from a capillary
microfluidic device according to aspects of the present
invention.
[0009] FIG. 1c provides a schematic illustration showing an
embodiment of the formation of a polymer microcapsule (MAMC) from a
W/O/W double emulsion according to aspects of the present
invention.
[0010] FIG. 2a displays a confocal midsection of isolated MAMCs
having inner aqueous cores containing fluorescently tagged
FITC-dextran and poly-lactic-co-glycolic acid (PLGA) shells tagged
with Nile Red according to aspects of the present invention; two
parameters of fabrication, MAMC shell thickness and the MAMC outer
diameter were investigated.
[0011] FIG. 2b illustrates two fabrication parameters, PLGA
concentration and fluid flow rates, affecting the MAMC shell
thickness to MAMC outer diameter ratio produced by a capillary
microfluidic device according to aspects of the present
invention.
[0012] FIG. 2c displays a table demonstrating the effects of PLGA
concentration and fluid flow rates on the characteristics of MAMCs
produced using a capillary microfluidics device according to
aspects of the present invention.
[0013] FIG. 2d displays confocal midsections of isolated MAMCs
produced by capillary microfluidics with varied parameters of
fabrication according to aspects of the present invention.
[0014] FIG. 3a illustrates a methodology to characterize
mechano-activation and load sensitivity using parallel plate
compression in which MAMCs are seeded between two cover slips and
compressed at a controlled increasing strain rate at a constant
temperature of 37.degree. C., according to aspects of the present
invention.
[0015] FIG. 3b illustrates mechano-activation and durability to
applied loads for three MAMC groups having different shell
thickness to MAMC outer diameter ratios or different MAMC outer
diameters according to aspects of the present invention.
[0016] FIG. 3c displays confocal microscopy images of MAMCs having
different different shell thickness to MAMC outer diameter ratios
or different MAMC outer diameters after applying increasing strain
rates according to aspects of the present invention.
[0017] FIG. 4a depicts a schematic of a methodology to characterize
degradation and durability of MAMCs having PLGA shells over 14 days
at constant physiologic temperature (37.degree. C.), according to
aspects of the present invention.
[0018] FIG. 4b illustrates mechano-activation of MAMCs having PLGA
shells incubated at a constant physiologic temperature of
37.degree. C. and subjected to different loads over 7 days,
according to aspects of the present invention.
[0019] FIG. 4c displays confocal microscopy images of MAMCs having
PLGA shells subjected to different loads after incubation at
37.degree. C. at days 1, 3, and 7, according to aspects of the
present invention.
[0020] FIG. 5a illustrates mechano-activation profiles of three
different groups of MAMCs after seven days of incubation at
37.degree. C., according to aspects of the present invention.
[0021] FIG. 5b illustrates mechano-activation profiles of three
different groups of MAMCs after fourteen days of incubation at
37.degree. C., according to aspects of the present invention.
[0022] FIG. 6 displays confocal microscopy and scanning electron
microscropy images of MAMCs after seven days of incubation at
37.degree. C., according to aspects of the present invention.
[0023] FIG. 7a illustrates a device for imaging mechano-activation
and deformation of MAMCs in a three-dimensional (3D) gel matrix,
according to aspects of the present invention.
[0024] FIG. 7b illustrates quantification of deformation, or aspect
ratio (AR), of MAMCs in 3D gel matrix, as a function of applied
strain, according to aspects of the present invention.
[0025] FIG. 7c depicts microscopy images of MAMCs in 3D gel matrix
according to aspects of the present invention before and after
injurious compression.
[0026] FIG. 7d depicts microscopy images of MAMC deformation of
MAMCs in 3D gel matrix during stepwise deformation of the gel
according to aspects of the present invention.
[0027] FIG. 8 illustrates field emission microscopy (FEM) of an
embodiment of a gel-MAMC composite showing von Mises stress with
gel deformation according to aspects of the present invention.
[0028] FIG. 9a illustrates strain and deformation of MAMCs in two
different gel matrices having different stiffness levels (50 kPa
and 500 kPa), according to aspects of the present invention.
[0029] FIG. 9b depicts confocal microscopy images of MAMCs
demonstrating levels of deformation in two different gel matrices
having different stiffness levels (50 kPa and 500 kPa), according
to aspects of the present invention.
[0030] FIG. 9c illustrates a methodology to characterize and
quantify mechano-activation and strain on MAMCs embedded in PEGDA
gel matrix, according to aspects of the present invention.
[0031] FIG. 10 illustrates active TGF-.beta.3 release from ruptured
MAMCs having poly(lactic-co-glycolic)acid shells embedded in
hydrogel matrix, according to aspects of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0032] Embodiments of the present invention relate to
mechanically-activated microcapsules (MAMCs) for controlled
drug-delivery, wherein the MAMCs release one or more active
ingredients in response to mechanical stimuli. According to
particular embodiments, the MAMCs of the present invention have
release profiles that are tuned to mechanical loading as a
mechanism for controlled drug delivery. The MAMCs provide a
platform for stimulating biological regeneration and repair in
mechanically-loaded tissues of a subject's body (e.g.,
musculoskeletal tissues, cartilage tissue, etc.).
[0033] Previous microcapsules have included various triggered
release mechanisms involving chemical or thermal triggers for drug
delivery (e.g., pH, heat, osmotic swelling, etc.). However,
mechanical feedback has played little role, if any, in release of
the active ingredient from these microcapsules. There are currently
no microcapsule delivery systems with release mechanisms that are
tuned to the endogenous mechanical environment. Embodiments of the
MAMCs of the present invention are not designed to rupture (and
preferably do not rupture) in response to non-mechanical
environmental cues including, without limitation, chemical or
thermal triggers, such as a change in pH, or an application of
light, heat, osmotic swelling, magnetic field, or ultrasound.
[0034] As used herein, mechanically-activated microcapsules (MAMCs)
of the present invention (also referred to herein as
"microcapsules") comprise hollow microcapsules that encapsulate one
or more active ingredients inside a solid shell. According to
particular embodiments, the solid shell comprises one or more
polymers (i.e., the shell is a "polymer shell"); preferably, the
shell comprises poly(lactic-co-glycolic)acid (PLGA) or another
FDA-approved material. When pressure/stress/deformation is applied
to the MAMCs (e.g., when the MAMCs are compressed), the solid shell
begins to deform.
[0035] When enough pressure (or "load") is applied, the MAMC
ruptures, which means that the solid shell breaks and the active
ingredient is released from the core of the MAMC into the
surrounding environment. Thus, the shell of the microcapsule breaks
when a minimum pressure threshold is applied.
[0036] The active ingredient(s) are preferably suspended in an
aqueous phase that is completely surrounded by the solid shell. As
used herein, the "active ingredient" (also referred to as
"therapeutic," "active pharmaceutical ingredient," "API," "drug,"
"biologic," or "active") refers to the pharmaceutically active
compound(s) encased inside the core defined by the solid shell.
According to particular embodiments, the active ingredient(s) are
selected from the group consisting of one or more anti-catabolic
compounds that inhibit or arrest cartilage breakdown (e.g.,
doxycycline), one or more anabolic compounds that encourage repair
and regeneration of cartilage (e.g., transforming growth factor
beta (TGF-.beta.)), one or more anti-inflammatory compounds, and
combinations thereof.
[0037] According to preferred embodiments, when MAMCs are delivered
to a region of a subject's body (e.g., a joint), the amount of
active ingredient(s) released from the MAMCs is a therapeutically
effective amount, i.e., the active ingredient(s) that are released
from the MAMCs upon rupture of the shell will have a desired
therapeutic effect within that region of the subject's body,
depending on the nature or severity of that subject's disease or
condition; for example, an amount of active ingredient(s) which
will cure, prevent, inhibit, or at least partially arrest, delay
the onset of or partially prevent a target disease or condition
(e.g., tissue damage, tissue breakdown, or tissue inflammation,
such as cartilage legions, cartilage injury, or cartilage breakdown
within a joint), or one or more symptoms thereof. The embodiment
includes delivery to native tissue such as into a joint after
injury, or as part of a tissue repair modality (i.e., defect repair
with a tissue engineered construct or other cell-based or acellular
(non-cell based) therapeutic). Anti-catabolic compounds, anabolic
compounds, and anti-inflammatory compounds are well-known, and
those of ordinary skill in the art can readily determine
appropriate dosages and amounts for use in accordance with the
present invention. The terms "subject" and "patient" are used
interchangeably herein and refer to a mammalian individual,
preferably a human being or animal (e.g. a domesticated pet or
thoroughbred horse).
[0038] According to particular embodiments, the
mechanically-activated microcapsules of the present invention have
diameters ranging from about 0.5 .mu.m to about 300 .mu.m, or about
1 .mu.m to about 300 .mu.m, or about 5 .mu.m to about 300 .mu.m, or
about 10 .mu.m to about 300 .mu.m, or about 0.5 .mu.m to about 200
.mu.m, or about 1 .mu.m to about 200 .mu.m, or about 5 .mu.m to
about 200 .mu.m, or about 10 .mu.m to about 200 .mu.m, or about 0.5
.mu.m to about 100 .mu.m, or about 1 .mu.m to about 100 .mu.m, or
about 5 .mu.m to about 100 .mu.m, or about 10 .mu.m to about 100
.mu.m, or about 20 .mu.m to about 300 .mu.m, or about 20 .mu.m to
about 200 .mu.m, or about 20 .mu.m to about 100 .mu.m, or about 20
.mu.m to about 75 .mu.m. Preferably, the MAMCs have diameters of
about 30 .mu.m to about 70 .mu.m, or about 40 .mu.m to about 60
.mu.m. According to particular embodiments, the shells of the
mechanically-activated microcapsules have a thickness of between
about 0.05 .mu.m to about 30 .mu.m, or between about 0.05 .mu.m to
about 20 .mu.m, or between about 0.05 .mu.m to about 10 .mu.m, or
between about 0.05 .mu.m to about 5 .mu.m, or between about 0.1
.mu.m to about 30 .mu.m, or between about 0.1 .mu.m to about 20
.mu.m, or between about 0.1 .mu.m to about 10 .mu.m, or between
about 0.1 .mu.m to about 5 .mu.m, or between about 0.25 .mu.m to
about 5 .mu.m, or between about 0.25 .mu.m to about 4 .mu.m, or
between about 0.25 .mu.m to about 3 .mu.m, or between about 0.5
.mu.m to about 5 .mu.m, or between about 0.5 .mu.m to about 4
.mu.m, or between about 0.5 .mu.m to about 3 .mu.m , or between
about 0.5 .mu.m to about 2.5 .mu.m.
[0039] According to particular embodiments, the MAMCs of the
present invention are made according to the methods described in
the 2012 publication by Fuquan Tu and Daeyeon Lee, Controlling the
Stability and Size of Double-Emulsion-Templated
Poly(lactic-co-glycolic) Acid Microcapsules; Langmuir, 2012;
28(26): pp. 9944-9952, which is incorporated by reference herein,
in its entirety and for all purposes. As described therein,
microfluidic techniques are used to generate water-in-oil-in-water
(W/O/W) double emulsions, which are used to template microcapsule
formation. Water-in-oil-in-water (W/O/W) double emulsions are
preferably generated using a glass capillary microfluidic device
(e.g., FIGS. 1a and 1b). To generate double emulsions, three
different fluid phases are injected into the microfluidic device by
three syringe pumps with controlled flow rates. According to
preferred embodiments, a glass capillary microfluidics device (see,
e.g., FIG. 1b) is utilized, wherein the inner phase comprises water
and drug(s), the middle phase comprises one or more polymers (e.g.,
PLGA) and optionally one or more plasticizers and/or one or more
stabilizers, and the outer phase comprises one or more stabilizers
(e.g., polyvinyl alcohol, surfactant(s), etc.). See FIG. 1c.
[0040] Embodiments of the present invention utilize
mechanically-loaded environments (e.g., a subject's joint) to
trigger and control release of therapeutics. Upon rupture, one or
more active ingredients entrapped within the microcapsules can
stimulate (i) anabolic processes leading to cell proliferation,
differentiation, and/or matrix biosynthesis, or (ii) anti-catabolic
processes that inhibit or arrest cartilage breakdown, or (iii) a
host of other responses. Because the timing of release is
controlled by mechanical load, the release of therapeutics can be
"tuned" based on the mechano-sensitivity of the microcapsules. For
example, mechanical activation of the microcapsules can be tuned
through material selection and microcapsule design, thereby
enabling the delivery of encapsulated active ingredients in
response to mechanical loading (e.g., walking, running, high-impact
sports, etc.). The release properties of the MAMCs under different
mechanical loading scenarios can be controlled, for example, by
modifying shell thickness-to-radius ratio and shell
elasticity/plasticity. Additionally, the composition of the shell,
and its degradation in a physiologic setting (e.g., the knee joint)
can be further used to define release profiles with expected
loading scenarios during rehabilitation and functional tissue
formation.
[0041] Tuning MAMC rupture characteristics enables the release of
active ingredients that stimulate repair and healing in an
"on-demand" fashion, thereby providing specific, local, and
controlled delivery. For example, MAMCs of the present invention
may be embedded within engineered matrices (e.g., engineered
cartilage) and may foster regeneration via controlled loading
during rehabilitation, or the MAMCs may be designed to actuate in
response to injury that occurs during supra-physiologic loads, so
as to promote rapid local repair after injury. Embodiments of this
technology have the potential to transform the treatment of
cartilage lesions and other mechanically-loaded tissues by
fostering maturation, preventing degeneration, and/or inducing
healing upon tissue damage through the delivery of therapeutics
with mechanical perturbation. Thus, the MAMCs can provide a broad
range of potential applications in directing regeneration in
mechanically-loaded tissues.
[0042] A microcapsule's minimum pressure/deformation/stress
threshold (i.e., the amount of pressure that is needed to break its
shell) varies depending on certain characteristics of the
microcapsule (e.g., mechanical properties and structure of the
microcapsule shell). In accordance with embodiments of the present
invention, characteristics of the microcapsule shell can be "tuned"
(i.e., optimized or adjusted) to provide a specific rupture profile
based on the mechanical loads that are expected to be applied to
the microcapsules after they have been administered to a subject. A
"rupture profile" (or "release profile" or "rupture
characteristics") refers to the minimum mechanical threshold that
must be applied to the microcapsule in order to rupture its shell
so that contents encased inside the core of the microcapsule (e.g.,
one or more APIs) can be released into the surrounding environment.
For example, after the microcapsules have been injected or embedded
into a subject's joint or other tissue, the drug is released from
the microcapsules when the minimum threshold
pressure/stress/deformation is realized in the joint, thereby
rupturing the microcapsules' shells.
[0043] According to particular embodiments, the microcapsules are
designed to have specific rupture profiles that enable the shells
to rupture and release the active ingredient(s) when subjected to
one or more specific mechanical loads within a subject's body
(e.g., within a subject's joint). Thus, the microcapsules' shells
are designed to rupture when one or more expected mechanical loads
are applied to the microcapsules. For example, when microcapsules
are administered into a subject's joint, the rupture profiles
correlate with the expected mechanical loads that will occur in the
joint so that when those mechanical loads are applied to the
microcapsules, the microcapsules rupture and release the active
ingredients. This allows for controlled drug delivery in response
to specific mechanical stimuli or mechanical perturbation. An
"expected mechanical load" is the amount of pressure that is
expected to be applied in a specific region of a subject's body
(e.g., a joint, such a knee joint, elbow joint, shoulder joint,
ankle joint, hip joint, or wrist joint) based on mechanics of that
region that are known in the art. According to additional
embodiments, the MAMCs can be delivered to a region of the body
that is not a joint; for example, the MAMCs can be delivered to
non-joint tissue (i.e., tissue that is not located within a joint),
such as skin, tendon or other tissue, to promote healing or control
growth or respond to injury.
[0044] The microcapsules may also be designed to break at different
loads depending on the type of therapy they are intended to deliver
to the subject. For example, the microcapsules may be intended for
prophylactic treatment after joint surgery, or for the prevention
of post-traumatic osteoarthritis, of for improving cartilage
regeneration and repair, or for the delivery of anti-inflammatory
therapeutic(s) during high-impact sports. If microcapsules are
delivered to a subject's knee, it may be desirable for at least
some of the microcapsules to rupture in response to relatively
"lower impact" movements (i.e., movements that place relatively
lower mechanical loads on the knee), such as walking, and/or for at
least some of the microcapsules to rupture in response to "higher
impact" movements (i.e., movements that place relatively higher
mechanical loads on the knee) such as deep flexion beyond
90.degree. or squatting or movements that are controlled for
therapeutic purposes (e.g., during use of post-surgery passive
motion devices). The microcapsules may be designed to rupture after
a single event (e.g., after exposure to a single mechanical load),
or after multiple events over time (e.g., after multiple exposures
to mechanical loads). The timing of degradation of the MAMC may
also be tuned to elicit the appropriate release profile during
rehabilitation or in response to injury.
[0045] According to an aspect of the present invention, the
mechanically-activated microcapsules that are administered to a
subject have one rupture profile, i.e., all of the MAMCs have the
same rupture profile or substantially the same rupture profile.
According to another aspect of the present invention, the
mechanically-activated microcapsules that are administered to a
subject have more than one rupture profile, i.e., the MAMCs have
varying rupture profiles such that some of the MAMCs have higher
minimum pressure thresholds than other MAMCs. This embodiment
allows for sequential delivery of one or more therapeutics when
microcapsules with different load thresholds are combined together
in a region of the subject's body. This embodiment also may allow
for continual release as the regenerate tissue that is forming
develops increasing mechanical properties, allowing for greater
stress transfer to the MAMC as the tissue matures in situ.
[0046] According to an embodiment of the present invention, a
method of tuning the rupture profiles of mechanically-activated
microcapsules to deliver a therapy to a subject comprises
identifying one or more "target" rupture profiles that will provide
a desired therapy to a subject based on mechanical loads that are
expected to be applied to mechanically-activated microcapsules
after they have been administered to a region of the subject's body
(e.g., by determining or estimating the mechanical load to which
the region of the subject's body is subjected), and creating
mechanically-activated microcapsules that have the one or more
target rupture profiles, wherein the mechanically-activated
microcapsules are designed to rupture and release a therapeutically
effective amount of one or more active ingredients (i.e., an amount
effective to provide the desired therapy) when the expected
mechanical load(s) are applied to the mechanically-activated
microcapsules. The MAMCs having the appropriately tuned rupture
profiles can then be delivered to the desired region of the
subject's body for the therapy.
[0047] According to particular embodiments, mechanically-activated
microcapsules having the target rupture profile(s) can be created
by providing a suitable shell thickness or mechanical property of
the shell. For example, MAMCs with a relatively smaller shell
thickness (e.g., 0.5 microns) have a lower minimum pressure
threshold that must be applied to rupture the shell, compared to
MAMCs with a relatively larger shell thickness (e.g., 2.5 microns),
which will have a higher minimum pressure threshold that must be
applied to rupture the shell (assuming all the other
characteristics of the MAMCs are the same). Shell thickness can be
modified, for example, by (i) changing the flow rate of at least
one fluid injected into the microfluidic device selected from
middle and inner fluids, and/or (ii) changing the ratio of flow
rates of the middle fluid and inner fluid injected into the
microfluidic device, and/or (iii) changing the concentration of
PLGA in the middle phase.
[0048] According to additional embodiments, the rupture profile(s)
can be controlled by modifying mechanical properties of the
mechanically-activated microcapsule shells, for example, by
modifying the stiffness or ductility of the shells. Stiffness and
or ductility and/or durability may be modified by modifying the
concentration of one or more plasticizers in the shells (e.g., by
adding one or more plasticizers to the shell compositions). The one
or more plasticizers may be selected from the group consisting of
diethyl phthalate, tributyl acetyl citrate, vitamins, vegetable
oils and a combination thereof. According to particular
embodiments, the higher the concentration of plasticizer(s) in the
shell, the higher the minimum pressure threshold that must be
applied to rupture the shell.
[0049] According to an embodiment of the present invention, a
method of using mechanically-activated microcapsules for drug
delivery comprises delivering mechanically-activated microcapsules
(MAMCs) to a region of a subject's body, wherein the
mechanically-activated microcapsules have one or more rupture
profiles that enable the mechanically-activated microcapsules to
rupture in response to one or more mechanical loads in said region
of the subject's body. Preferably, the MAMCs are delivered to a
region of a subject's body that is a joint, most preferably an
articulated joint (e.g., a knee joint, elbow joint, shoulder joint,
wrist joint, ankle joint or hip joint). Alternatively, the MAMCs
can be delivered to a "non-joint" region of the subject's body
(e.g., skin). The MACMs are preferably designed to provide a
localized therapeutic effect to the region of the subject's body in
which they are delivered (as opposed to a systemic therapeutic
effect).
[0050] According to one aspect of the invention,
mechanically-activated microcapsules are injected or otherwise
administered into a subject's joint. For example, the microcapsules
are injected into tissue that is within or surrounding the joint
(e.g., the microcapsules are injected into musculoskeletal tissue
or cartilage located in or around the joint). According to another
aspect of the invention, the mechanically-activated microcapsules
are embedded within a matrix material (e.g., gel and/or engineered
tissue for cartilage repair/replacement) and the matrix material is
delivered into a subject's joint. For example, the MAMCs may be
embedded in a polymeric material that has a stiffness comparable to
native cartilage; the polymeric material may comprise poly(ethylene
glycol) diacrylate (PEGDA), methacrylated hyaluronic acid (HA), or
one or more other polymers. The matrix material can be injected
into a joint or implanted surgically into a joint.
[0051] According to particular embodiments, the MAMCs that are
delivered to a region of the subject's body have a plurality of
different rupture profiles (e.g., at least two different minimum
load thresholds). For example, the MAMCs have more than one shell
thickness (e.g., selected from 0.5, 1, 1.5, 2, and 2.5 microns)
and/or one or more shell compositions (e.g., selected from PLGA
without any plasticizers, PLGA with one or more plasticizers, and
PLGA with varying concentrations of plasticizer). The MAMCs having
a plurality of different rupture profiles (e.g., two or more
rupture profiles) may be embedded into a matrix material that is
implanted into a joint. A mixture of MAMCs with varying rupture
profiles may be desirable for rehabilitation regimens in which the
active ingredients are released from embedded microcapsules as the
implanted matrix material degrades over time. The method may
further comprise a step of selecting the mechanically-activated
microcapsules according to their rupture profile(s).
[0052] According to embodiments of the invention in which the MAMCs
are embedded within a matrix (e.g., a polymer material), the
rupture profile of the MAMCs can be adjusted by modifying the
physical properties of the matrix, thereby modifying the adhesion
of the MAMCs to the matrix. Strong interfacial adhesion between
microcapsules and the matrix is known to effect capsule rupture.
Thus, modifying the nature of adhesive interaction (e.g.,
electrostatic vs. protein-ligand interactions) can influence
rupture. Also, modifying the ratio of the elastic modulus of
embedding matrix to that of shell may determine whether crack
propagation occurs through or deflects around microcapsules. During
fabrication of MAMCs, adhesion properties can be tuned; for
example, by covalently bonding microcapsules to the hydrogel upon
photopolymerization. Alternatively, adhesion can be controlled
using electrostatic interactions where either the shell or hydrogel
is modified with a surface charge. Thus, there are several ways in
which the MAMCs can be designed to rupture in response to the
mechanics or degeneration of a matrix in which they are
embedded.
[0053] According to an embodiment of the present invention, a
composition comprises a mixture of MAMCs having a plurality of
rupture profiles. For example, the MAMCs in the composition have
more than one shell thickness (e.g., selected from 0.5, 1, 1.5, 2,
and 2.5 microns) and/or one or more shell composition (e.g.,
selected from PLGA without any plasticizers, PLGA with one or more
plasticizers, and PLGA with varying concentrations of plasticizer).
The composition may further comprise a pharmaceutically acceptable
carrier (i.e., an excipient, diluent, preservative, solubilizer,
emulsifier, adjuvant, and/or vehicle with which the MAMCs are
administered). For example, such carriers may comprise a liquid,
such as water, saline solution, dextrose solution, fibrin gel, or
glycerol solution. The composition may also comprise one or more
excipients (e.g., wetting or emulsifying agents; pH buffering
agents such as acetates, citrates or phosphates; antibacterial
agents such as benzyl alcohol or methyl parabens; antioxidants such
as ascorbic acid or sodium bisulfite; chelating agents such as
EDTA, etc.). According to particular embodiments, the composition
is embedded within a matrix (e.g., a hydrogel or other polymeric
material that does or does not deliver cells and that has a
stiffness similar to that of native cartilage or that can mature to
that point via cell-mediated matrix deposition).
[0054] According to particular embodiments, a method of making a
composition of the present invention comprises mixing the
components of the composition together (e.g., mixing the MAMCs
having a plurality of rupture profiles with an optional
pharmaceutical carrier and one or more optional excipients). The
method may further comprise embedding the composition within a
matrix material. The compositions of the present invention can be
administered to a subject in accordance with any of the methods
described herein; for example, by injection into a subject's
joint.
[0055] As described herein, mechanically activatable microcapsules
can be used to controllably deliver bioactive factors upon
mechanical stimulation. This has specific applications in cartilage
repair, where rehabilitation regimens might be tuned to slowly
instigate release of factors from embedded microcapsules as the
implanted tissue matures. In addition, this technology can be
applied to instances of cartilage injury due to trauma. Inclusion
of MAMCs that release in response to injury may be used to protect
regenerate tissue and improve durability of repair in populations
at risk for re-injury. More generally, this technology may find
broad application in a variety of mechanically loaded
musculoskeletal regeneration and repair applications.
[0056] The embodiments of the invention are described above using
the term "comprising" and variations thereof. However, it is the
intent of the inventors that the term "comprising" may be
substituted in any of the embodiments described herein with
"consisting of" and "consisting essentially of" without departing
from the scope of the invention. Unless specified otherwise, all
values provided herein include up to and including the starting
points and end points given.
[0057] The following examples further illustrate embodiments of the
invention and are to be construed as illustrative and not in
limitation thereof.
EXAMPLES
Example 1
[0058] Fabrication of mechanically-activated microcapsules (MAMCs)
and determination of how variations in fabrication parameters
influence the structure-release properties of individual MAMCs.
[0059] In this first formulation, microcapsules were 100 .mu.m in
diameter with a shell thickness of 1 .mu.m, and the shell was doped
with a fluorescent dye (Nile Red) to enable visualization (FIG.
2a). To demonstrate mechano-activation, a single layer of MAMCs was
subjected to increasing levels of load using a mechanical testing
device (FIG. 2a). Results showed graded microcapsule rupture and
release of FITC-dextran with increasing load (FIG. 2b). Intact
microcapsules served as negative controls and sheared microcapsules
(completely devoid of FITC-dextran due to complete rupture) served
as positive controls. Fluorescent intensity of the buffer solution
(indicating FITC-dextran release) correlated with load. Similar
activity assays can be used to directly measure cumulative drug
release for a given application.
[0060] In this example, a model microcapsule system comprised a
poly(lactic-co-glycolic) acid (PLGA) copolymer shell with a soluble
fluorescently-tagged molecule (FITC-dextran) encapsulated within an
aqueous core. The MAMCs were fabricated using a glass-capillary
microfluidic system (FIGS. 1a and 1b) to produce a highly
monodisperse water-in-oil-in-water (W/O/W) emulsion with
approximately 100% encapsulation efficiency (FIG. 1c). Physical
characteristics of the MAMCs were modified, including shell
thickness-to-outer diameter ratio, to define thresholds at which
different MAMCs fail and release entrapped biomolecules. A
systematic approach can be used to develop a suite of MAMCs with
different properties, and their elastic, plastic, and failure
behaviors may be assessed using quantitative micromechanical
analysis tools (including atomic force microscopy (AFM) and
reflective interference contrast microscopy). This example may
further define the mechanical thresholds that instigate
microcapsule rupture, and may further establish the parameter space
under which these MAMCs are operative.
[0061] 1. Microcapsule Fabrication.
[0062] As described above, a glass-capillary microfluidic device
(FIGS. 1a and 1b) were used to fabricate MAMCs with differing
physical attributes. Double emulsions were formed having inner
aqueous phases containing a "model" drug (e.g., FITC-dextran, which
is a fluorescently tagged molecule). The aqueous solution including
the model drug was, in this instance, saline. The middle fluid
phase of the double emulsion, or the oil phase, included a suitable
polymer for producing the shell of the MAMCs (e.g.,
poly-lactic-co-glycolic acid or PLGA). The polymer may be
biodegradable and biocompatible. The oil phase including the
polymer was, in this instance, chloroform. The oil phase was also
be doped with a dye compound (e.g., Nile Red) to visualize the
resulting MAMC shells. The outer fluid phase was also an aqueous
fluid to result in a W/O/W double emulsion (FIG. 1c). The final
result of the fabrication was MAMCs having Nile Red-dyed PLGA
shells encapsulating fluorescent FITC-Dextran. FIG. 2a displays a
confocal midsection of MAMCs produced according to this process.
The inventors hypothesized that the ratio (t/D) between two
parameters of fabrication, the thickness of the microcapsule shell
and the outer diameter of the microcapsule, would control release
thresholds and mechano-activation of the microcapsules. See FIG.
2a.
[0063] MAMC Shell Thickness.
[0064] Shell thickness can be varied by altering the polymer (e.g.,
PLGA) concentration in the oil phase of the double emulsion, or by
changing the ratio of flow rates of the middle oil phase (e.g.,
containing PLGA) and inner aqueous phase (e.g., containing model
drug FITC-dextran). See FIG. 2b. Increases in the polymer (e.g.,
PLGA) concentration increased the shell thickness, which increased
the shell thickness to outer microcapsule diameter ratio (t/D).
Decreasing the ratio of middle oil phase:inner aqueous phase flow
rates results in decreasing shell thickness; however, there is
often a lower limit for shell thickness due to susceptibility to
rupture in the high shear microfluidic environment, depending on
the radius of the microcapsule. Also, decreases in both flow rates
resulted in thinner shell walls and smaller microcapsule outer
diameter. See FIGS. 2c and 2d, which demonstrate the effects of
changing polymer concentration and flow rates on the t/D ratio.
Alternatively, changing the concentration of PLGA in the middle
phase may also affect shell thickness. Practical considerations for
optimizing shell thickness may include sensitivity to handling as
well as stiffness and proclivity to fracture under defined loads.
Target shell thicknesses may preferably range from about 0.5 .mu.m
to about 2.5 .mu.m.
[0065] Mechano-Activation of MAMCs with Different Dimensions.
[0066] The inventors hypothesized that the t/D ratios and outer
diameter fabrication parameters of the microcapsules affected the
mechano-activation of the microcapsules. A series of quantitative
assays were performed to evaluate MAMC properties as a function of
fabrication parameters. Atomic force microscopy (AFM) is a powerful
method for characterizing elastic and plastic deformation of single
microcapsules. For uniform compression, a tipless cantilever
(simulating a parallel plate) is used to load and unload MAMCs at a
constant displacement rate to generate force vs. displacement
curves. The linear region of these curves can be used to estimate
elastic properties of the shell, namely the shell stiffness (slope)
and the Young's modulus using an analytical solution. Thin shell
theory holds if: (1) the ratio of shell thickness to radius is less
than 1/20, (2) the shell material is linearly elastic, homogeneous,
and isotropic, (3) deformation is small (on the order of shell
thickness), and (4) shell thickness is constant. For large
deformations in the non-linear region of the force-deformation
curve, onset of permanent plastic deformations can be quantified by
measuring instability (microcapsule buckling) and hysteresis. With
repeated loading-unloading cycles (simulating fatigue),
instabilities may appear as inflection points at lower forces and
deformations. In addition, hysteresis may increase, such that the
traces no longer align. To extract intrinsic properties such as
yield and strain at rupture, finite element models may need to
simulate large deformations using 3D constitutive laws. Since thin
shell theory holds regardless of length scale, several trends have
been noted for predicting elastic and plastic behaviors of
microcapsules. (1) As diameter of the microsphere increases,
effective shell modulus decreases (approaches bulk properties) and
as shell thickness decreases, effective shell modulus increases and
is higher than the bulk polymer modulus. (2) For large
deformations, with increasing cantilever stiffness more
instabilities appear in smaller diameter microcapsules due to
higher forces. The probability of instability also increases with
more compliant shells. During characterization of MAMCs, single
cycle and repeated cyclic compression by AFM will be used (coupled
with measurement of fluorescent intensity of the microcapsule
contents) to establish deformation magnitude and cycle number
thresholds for failure for different MAMC formulations.
[0067] Another quantitative assay for characterizing
mechano-activation and load-sensitivity in MAMCs having different
fabrication characteristics was carried out using parallel plate
compression in which MAMCs were seeded between two glass coverslips
(FIG. 3a). The MAMCs were uniaxially compressed at a controlled
strain rate to specified loads ranging from 0 to 1 newton (N), with
5N serving as a positive, completely ruptured control. The MAMCs
were then collected and imaged using confocal microscopy to
quantify the fraction of empty microcapsules (FIG. 3c). Increasing
the shell thickness of the MAMCs resulted in increasing
insensitivity to load within the same load range (as demonstrated
by MAMCs of the same diameter, but different t/D ratios). See FIGS.
3b and 3c. However, the groups of MAMCs having the same t/D ratios,
regardless of outer MAMC diameters, demonstrated the same
mechano-sensitivity as load (N) increased. See FIGS. 3b and 3c. The
group of MAMCs having a t/D ratio of 0.009 (as a result of a
thicker outer shell) demonstrated very little mechano-sensitivity
from 0 to 1 newton. See FIGS. 3b and 3c. Thus, the inventors have
discovered that MAMC release profiles are highly dependent on the
t/D ratios, where the ratio and not only the shell thickness
influences crack propensity and durability to an applied load.
[0068] Mechanical properties of MAMC shell. In addition to
microcapsule dimensions, the inventors hypothesized that the
mechanical properties of the shell material influence rupture.
Rupture of single MAMCs may occur either (1) within or close to the
elastic regime (fractional deformation at rupture <0.2), or (2)
after extensive plastic deformation, depending on the brittle or
ductile nature of the polymer. Pure PLGA has an elastic modulus of
about 2-3 GPa. The PLGA shell can be doped with a biocompatible,
hydrophobic plasticizer (e.g., diethyl phthalate, tributyl acetyl
citrate) to reduce the shell stiffness. This low water-soluble
plasticizer, at appropriate concentrations (<10% w/v), will
increase plasticity of the shell, but still allow for rupture. In a
single cycle loading scenario (e.g., impact injury), brittle
failure would be preferable for immediate release, but for a
dynamic (cyclic) loading scenario (e.g., during rehabilitation of a
maturing engineered cartilage defect, over which cycles of loaded
and unloaded periods may occur, simulating fatigue), plasticity of
the microcapsule will be particularly important for regulating
delayed onset of rupture. Different ranges of plasticizer
concentrations may manipulate MAMC elastic and plastic deformation
characteristics to tune release; for example, plasticizer
concentrations may be varied between about 0.01 wt % and about 30
wt % plasticizer relative to the total shell composition to change
release characteristics.
[0069] Reflective Interference Contrast Microscopy (RICM) is used
to measure the adhesion area and energy of microcapsules on a
specific substrate (to simulate matrix-microcapsule interactions).
Microcapsules are illuminated with monochromatic light in a
reflection geometry and the interference pattern yields information
on the local distance between the capsule wall and glass surface
(post-processing image analysis of adhesion area). Adhesion is
influenced by the microcapsule-substrate interaction and by the
properties and geometry of the shell, where softer microcapsules
with a low thickness-to-radius ratio have higher adhesion energy.
Using the above techniques, a range of shell thicknesses and
polymer compositions can be assayed to determine how these
parameters influence MAMC deformation and rupture under static and
dynamic load.
The inventors' discoveries regarding the fabrication parameters of
MAMCs and shell mechanical properties allow for a suite of
microcapsules to be developed whose mechano-activation is defined
by the mechanical properties and dimensions of the shell and
overall microcapsule. Different formulations may be developed based
on different MAMC characteristics, for example, variations in shell
thicknesses (e.g., 0.5, 1, 1.5, 2, and 2.5 microns), outer
diameters of the microcapsules, and material properties of the
shell (e.g., PLGA, PLGA with low plasticizer, PLGA with high
plasticizer). Thus, activation of these MAMCs may be achieved
across a range of loads/deformations and number of compression
cycles. This may allow for tuning of release in response to forces
and deformations experienced by the MAMCs in the model gel systems
and in living constructs exposed to physiologic and
pathophysiologic loading.
Example 2
[0070] In this example, the effects of polymer degradation of MAMC
shells at physiologic temperature (37.degree. C.) on
mechano-activation were explored. Using the same parallel plate
technique as described above in Example 1, MAMCs were tracked over
a period of 14 days at different loads between 0 and 1 Newton, with
5 Newtons as a completely ruptured control. See FIGS. 4a and 4b.
The group of MAMCs demonstrating the highest resistance to load
from Example 1 (t/D of 0.009 and outer diameter of 105 .mu.m) was
used. Within the first three days of incubation at 37.degree. C.,
the mechano-sensitivity of the MAMCs was unaffected. However, by
day 7, the microcapsule mechanical release profile is significantly
affected by application of load. See FIGS. 4b and 4c. This
demonstrates that degradation of the polymer shell has a marked
effect on mechanically controlled rupture and release of the
MAMCs.
[0071] A similar methodology was carried out over 14 days using
three different classes of MAMCs. See FIGS. 5a and 5b. The sum
effect of degradation demonstrated that the bulk erosion of shell
polymer (e.g., PLGA) resulted in the convergence of
mechano-activation profiles across a range of t/D ratios after
seven days of incubation at 37.degree. C. (FIG. 5a), but by day 14,
even microcapsules under zero load had lost their encapsulated
contents and mechanical integrity as quantified by the empty
fraction (FIG. 5b). By day 14, all microcapsules subjected to load
completely fractured.
[0072] FIG. 6 depicts reconstruction of the volume (confocal
microscopy images) and the morphology (scanning electron microscopy
images) of MAMCs having PLGA shells and core FITC-dextran contents,
after seven days of incubation at 37.degree. C. The images
demonstrate the rupture mechanism in three dimensions. Control
samples (under ON load) maintain their core FITC-dextran contents,
but the same MAMCs demonstrate loss of fluorescence under 1N load.
Differences in morphology were confirmed using scanning electron
microscopy, with MAMCs shown to be ruptured across the midplane of
the sphere under 1N load.
Example 3
Microcapsule Mechano-Activation in 3D Hydrogels.
[0073] In this example, the deformation of MAMCs embedded in 3D
matrices (analogous to engineered constructs mimicking cartilage)
was explored. To validate mechano-activation in a three-dimensional
(3D) construct, microcapsules were embedded in 30%
photo-crosslinked poly(ethylene glycol) diacrylate (PEGDA). See
FIG. 9c. This hydrogel was chosen because its stiffness is
comparable to native cartilage and matured engineered cartilage.
Using a custom micromechanical compression device mounted on a
confocal microscope (FIG. 7a), MAMC-laden hydrogels were compressed
in unconfined compression (0-20% strain, steps of 4%, followed by
compression until hydrogel failure). MAMCs deformed with increasing
hydrogel compression, becoming ellipsoid at 20% strain and visibly
rupturing at 60% strain (FIGS. 7b and 7d). FIG. 7b depicts the
performance of MAMCs embedded in 3D gel matrix, as a function of
applied strain; FIG. 7b further demonstrates how the surrounding
matrix stiffness controls when and whether a MAMC will deform and
rupture. To test for rupture under pathophysiologic loading,
injurious compression (20% strain at 50% strain/sec) was applied to
fracture the hydrogel. Here, MAMCs buckled and released their
contents (FIG. 7c).
[0074] In the following examples, further analysis of MAMC
deformation in 3D matrices and the mode of rupture (brittle vs.
ductile failure) as a function of fabrication parameters defines
mechanical thresholds and enables tunable release.
Example 4
[0075] Measuring and modeling rupture and biofactor release from
MAMCs embedded in 3D matrices as a function of matrix stiffness,
matrix-capsule adhesion, and loading.
[0076] In this example, microcapsule properties (e.g., the ratio of
shell elasticity to matrix elasticity, shell thickness, and
adhesion to local matrix) are studied with respect to how these
properties dictate failure and release characteristics with loading
when MAMCs are embedded in 3D matrices of varying properties. Using
a novel micromechanical test system, deformation and rupture of
MAMCs embedded in 3D matrices (photo-crosslinked hydrogels) of
varying elasticity are evaluated as a function of loading
conditions, and the release of encapsulated fluorescent molecules
will be tracked by confocal microscopy to determine mechanical
thresholds for each microcapsule formulation. Finite element (FE)
analysis are used to model microcapsule mechanical response, and
rupture and release will be predicted over a range of microcapsule
fabrication and 3D matrix properties. Microcapsule-gel composites
are evaluated through the following assays.
[0077] A similar methodology to that described in Example 3 was
carried out to model engineered cartilage by embedding
microcapsules in PEGDA hydrogel. The PEGDA hydrogel has mechanical
properties that can be tuned to mimic immature or mature engineered
cartilage. Uniaxial compression on a confocal mounted device (FIG.
7a) was applied to the MAMCs embedded in the hydrogel at increments
of 5% to 20% strain. See FIG. 9c. Microcapsule strain was
quantified in the direction of the uniaxial strain (E.sub.11) or
perpendicular to the direction of the uniaxial compression
(E.sub.22). See FIG. 9a. FIG. 8 is a diagram depicting field
emission microscopy (FEM) of an embodiment of a gel-MAMC composite
showing von Mises stress with gel deformation, and the strain
profile placed upon an embedded microcapsule as a result of uniform
compression
[0078] 1. Adhesion.
[0079] In conventional self-healing polymers, strong interfacial
adhesion between microcapsules and the matrix is critical for
promoting capsule rupture upon crack formation. A rough exterior
shell wall, decreasing capsule shell-to-radius ratio, and the
nature of adhesive interaction (e.g., electrostatic vs.
protein-ligand interactions) can influence rupture. For embedded
MAMCs, the ratio of the elastic modulus of embedding matrix to that
of shell may determine whether crack propagation occurs through or
deflects around microcapsules. During fabrication of MAMCs,
adhesion properties can be tuned. For immediate release, as in
cartilage repair after injury, the PLGA microcapsule shell will be
generated using a polymerizable or cross-linkable surfactant (e.g.,
pluronic diacrylate, as a non-limiting example) as a stabilizer in
double emulsion preparation. Pluronic diacrylate, if used, can be
synthesized using a previously reported method, and can covalently
bond microcapsules to the hydrogel upon photopolymerization.
Alternatively, adhesion can be controlled using electrostatic
interactions where either the shell or hydrogel is modified with a
surface charge (e.g., PLGA-g-PLL) with a positive charge attracted
to a negatively charged hydrogel. For delayed release scenarios
with pre-culture of engineered cartilage prior to
mechanically-induced release, it may be possible to rely on cell
adhesion and matrix deposition on a positively charged shell for
strong matrix adhesion. Optimizing this parameter may be important
for reliably predicting microcapsule rupture in various matrices
and loading environments, and may allow for tuning the rupture
sensitivity of MAMCs embedded in 3D matrices.
[0080] 2.Ratio of Shell:Matrix Elasticity.
[0081] To evaluate the rupture of microcapsules in 3D gels with
varying properties, MAMCs with defined shell moduli (based on AFM
results) can be embedded in different matrices (e.g., acellular
photo-crosslinked methacrylated hyaluronic acid (HA) or
poly(ethylene glycol) diacrylate (PEGDA) hydrogels). HA and PEGDA
hydrogels have been produced with compressive moduli spanning
5-2000 kPa. This allows for the ratio of the modulus of the shell
to that of the surrounding matrix to be altered across a wide
range. Previous models for autonomic self-healing materials (where
release of a catalyst from a microcapsule initiates polymerization)
have demonstrated that this ratio determines whether cracks are
deflected around (E.sub.shell=3E.sub.matrix) or directed to
microcapsules (E.sub.shell=1/3E.sub.matrix) to cause rupture. This
ratio influences the design of MAMCs targeted to rupture under
specific physiological loading (e.g., dynamic compressive loading
vs. singular compressive injury). MAMCs can be designed to rupture
across matrix moduli representative of engineered cartilage as it
matures (about 50 to 600 kPa).
[0082] In one methodology, microcapsules were embedded in two
different PEGDA hydrogel compositions having different levels of
stiffness (50 kPa versus 500 kPa). The two PEGDA hydrogel
composites have mechanical properties tuned to mimic immature
cartilage (50 kPa) and mature cartilage (500 kPa). See FIGS. 9a and
9b. Microcapsule strain was quantified in the direction of the
uniaxial strain (E.sub.11) or perpendicular to the direction of the
uniaxial compression (E.sub.22). Microcapsules embedded within the
softer matrix (50 kPa) experienced very little or no deformation
under increasing strain to the matrix, and even upon fracture the
shell of the microcapsules show only minimal deformation. In
contrast, microcapsules embedded in a matrix having a stiffness
ten-fold higher (500 kPa) deformed significantly over increasing
strain, with folds developing in the shell wall. The microcapsules
were permanently deformed upon hydrogel fracture as well. See FIGS.
9a and 9b. Additionally, previous experiments found that MAMCs
ruptured in 30% PEGDA hydrogels that had an elastic modulus of
about 1.9 MPa (about 2-fold stiffer than in vitro engineered
cartilage).
[0083] 3. Micromechanical Analyses and FE Modeling.
[0084] Deformation of MAMCs embedded in a hydrogel may be measured
using a custom compression device mounted on a confocal microscope
(FIG. 7a). Briefly, constructs are subjected to compression in a
step strain manner at intervals of 4% to a total of 20% strain,
with a hold of about 30 minutes between steps to allow for release
from ruptured MAMCs. Additional samples may be subjected to a
single loading event representative of a compressive injury. Image
stacks (.about.500 .mu.m) can be reconstructed to visualize MAMC
deformation and to determine deformation thresholds for release.
For each MAMC formulation, "dose-response curves" are constructed
by correlating applied strain to the fraction of ruptured MAMC. For
a given "physiologic" strain (i.e., 10%, used in long-term loading
studies), the same analysis is performed with cyclic deformation to
determine number of cycles before rupture (i.e., to demonstrate
rupture after fatigue). From these curves, loading conditions that
elicit rupture of 1/2 of the MAMCs for a given matrix elasticity
can be determined. To further this analysis, FE models of MAMC-gel
composites may be developed. While analytical solutions exist to
predict stress/strain in the matrix and the shell of a spherical
inclusion, FE models provide a versatile means by which to infer
the fracture behavior of MAMCs with variation in multiple
parameters. FE models can be built in Abaqus, and geometry,
boundary conditions (microcapsule-matrix adhesion), dimensions, and
material properties of shell and matrix explored. Deformation
behavior MAMCs at small strains (FIG. 8) can therefore be
predicted, and may guide the design of MAMCs with different rupture
behaviors with loading.
[0085] A series of MAMC/gel composites can therefore be generated,
demonstrating controlled release in response to different
mechanical perturbation (single compression injury, stepwise
compression, and/or dynamic loading or fatigue over cycles of
loading and unloading). By varying MAMC physical characteristics,
gel elasticity, and interaction of MAMCs with the gel, we can
identify and generate MAMC sets that rupture at different stages of
tissue maturation and in response to physiologic and
pathophysiologic loading.
Example 5
[0086] Demonstrating that biologics released from MAMCs can
stimulate maturation, repair, and/or disease control in response to
physiologic and pathophysiologic loading.
[0087] In this example, the effect of on-demand delivery of
biologics from MAMCs on altering the trajectory of tissue growth
and/or repair, or disease modification and/or control, in
engineered cartilage constructs is studied. MAMCs are first
embedded in hydrogel constructs and activated by loading to release
factors to promote tissue formation. Factors may be any biologics
or molecules capable of acting as therapeutics in
mechanically-loaded musculoskeletal tissues. These factors include,
but are not limited to, chondrogenic factors, anabolic compounds,
transforming growth factors (e.g., TGF-.beta., TGF-.alpha.),
fibroblast growth factors (e.g., FGF-2, FGF-18), connective tissue
growth factors (e.g., CTGF), insulin-like growth factors (e.g.,
IGF-1), and bone morphogenetic proteins (e.g., BMP-2, BMP-6). Other
factors include anti-catabolic, anti-inflammatory, or anti-cell
death compounds or biological therapeutics that may be used to
control or modify diseases in mechanically-loaded musculoskeletal
tissues.
[0088] This example can be used to specifically determine whether
load-induced release of transforming growth factor beta
(TGF-.beta.) is capable of instigating the chondrogenic
differentiation of mesenchymal stem cells (MSCs). TGF-.beta. is
also known to induce cartilage tissue formation and plays an
important role in cartilage repair and the repair of other
musculoskeletal tissues. Second, this may illustrate the
therapeutic potential of MAMCs tuned to release with
pathophysiologic loading, using a model of post-traumatic
osteoarthritis (PTOA). Embedded microcapsules in constructs can be
tuned to rupture upon injurious compression, and the effect of
local release of anti-catabolic and/or anabolic compounds may be
evaluated in terms of attenuation of matrix loss and reduction of
protease activity in the tissue.
[0089] 1. Programmed Release to Induce Chondrogenesis.
[0090] Engineered cartilage is formed by encapsulating MSCs in
photo crosslinked 1% HA hydrogels. In the absence of chondrogenic
factors, MSCs in HA gels do not differentiate or produce
cartilage-like matrix. Here, hydrogels include MAMCs having PLGA
shells loaded with the chondrogenic factor TGF-.beta.3. See FIG.
10. TGF-.beta.3 release from embedded microspheres (100 ng
TGF/construct) can induce chondrogenesis. For these studies, TGF
concentration in the core of the MAMCs may be defined so as to
place 100 ng per construct, based on the MAMC volume fraction (1%
v/v). Constructs with blank PLGA MAMCs (containing only PBS) are
used as negative controls. Medium supplemented with TGF (100
ng/construct, distributed evenly over each media change) may serve
as a positive control. The supernatant was analyzed by ELISA for
released TGF-.beta.3. Active TGF-.beta.3 release was observed from
ruptured MAMCs, with minimal leakage from non-ruptured MAMCs.
[0091] Based on Example 4, MAMCs may be selected to achieve
complete rupture, partial rupture, and no rupture over the course
of tissue maturation. From previous experiments, engineered
constructs cultured over 4 weeks achieve equilibrium moduli of
about 50-100 kPa, and dynamic moduli of about 500-1000 kPa. Based
on this, the inventors hypothesize that microcapsules with an
elastic modulus about 1/3 of that of the matrix are likely to
ensure complete release, while an elastic modulus approximately
equal to the matrix will result in partial release, and about
3-fold higher elastic modulus than the matrix will result in no
release. To verify release, cell-free HA gels of varying weight
percent (1-10% w/v), having equilibrium and dynamic properties
spanning the range of tissue maturation, are used. In cell-based
experiments, constructs are subjected to dynamic compression (10%
strain, 1 Hz, 3 hours per day, 5 days per week) to simulate
physiologic loading. Free-swelling constructs with embedded MAMCs
may serve as controls, with no microcapsule rupture expected to
occur. Constructs can be cultured for up to four weeks, with weekly
harvest (n=5) to evaluate MSC differentiation. Differentiation can
be assessed by Alcian blue staining of proteoglycan (PG) deposition
and measurement of biochemical content (PG and collagen) and
mechanical properties, as in previous studies. MAMC properties are
expected to correlate with growth factor release with load and
that, in cell-based constructs, release is expected to drive
chondrogenesis.
[0092] 2. Programmed Release after Injury.
[0093] An engineered cartilage model of injurious compression was
developed to investigate mechanisms of degeneration in PTOA using a
high throughput mechanical injury device for testing compressive
injury of engineered cartilage. Based on this model, chondrocytes
can be encapsulated in HA and pre-cultured for 8 weeks, followed by
injury. MAMCs are embedded in the constructs at the time of
formation. Here, MAMCs with elastic properties that are more
compliant than a mature construct (e.g., E.sub.hydrogel=300-500 kPa
at 8 weeks, E.sub.shell<<E.sub.hydrogel) can be used to guide
crack propagation through the microcapsules. Pre-matured constructs
are subjected to injurious compression (50% strain at 50%/s). After
injury, chondrocyte viability decreases and loss of matrix
increases. To determine whether MAMCs can alter these outcomes
following injury, two potential therapeutics are included during
MAMC fabrication: an anti-catabolic compound (MMP inhibitor
doxycycline) to reduce degradation, and an anabolic compound
(TGF-.beta.) to increase matrix biosynthesis. The effectiveness of
TGF-.beta.3 (10 ng/mL) in reducing GAG loss (by about 30% 5 days
post-injury) compared to injured controls has been shown. Low doses
of TGF-.beta.3 (10 ng/mL) and doxycycline (50ng/mL) also reduce MMP
activity after inflammatory stimulation of chondrocytes. Based on
this, the activity of these factors is first validated by testing
doses of 10 to 100 ng/construct. MAMCs are produced with factors at
these doses and constructs are injured and analyzed at 48 and 120
hours. Both un-injured constructs with embedded MAMCs and blank
MAMCs serve as controls. At each time point post injury, sulfated
glycosaminoglycan (s-GAG), present in the construct and released to
the media, is assayed (n=4). Additional samples may stained for
viability (Live/Dead) and proteoglycans (Alcian blue). In addition,
MMP inhibition can be evaluated by immunostaining for neo-epitopes
engendered by MMP activity (n=3) and active MMP can be assayed in
the media (Anaspec). As above, ELISA can be used to measure
TGF-.beta. release after injury.
[0094] The inventors hypothesize that MAMCs can be tuned to release
TGF-beta over the course of construct maturation for MSC-seeded
hydrogels, and this release caninduce chondrogenesis and matrix
accumulation. In the context of injurious compression, immediate
release from microcapsules is expected, enhancing cell viability
post-injury and attenuating loss of proteoglycan from
constructs.
[0095] The embodiments described herein are intended to be
exemplary of the invention and not limitations thereof. One skilled
in the art will appreciate that modifications to the embodiments
and examples of the present disclosure may be made without
departing from the scope of the present disclosure.
* * * * *