U.S. patent application number 17/009569 was filed with the patent office on 2021-02-04 for polymeric microneedles and rapid additive manufacturing of the same.
The applicant listed for this patent is The University of North Carolina at Chapel Hill. Invention is credited to Joseph M. Desimone, Ashley R. Johnson, Gregory R. Robbins.
Application Number | 20210031439 17/009569 |
Document ID | / |
Family ID | 1000005152404 |
Filed Date | 2021-02-04 |
View All Diagrams
United States Patent
Application |
20210031439 |
Kind Code |
A1 |
Desimone; Joseph M. ; et
al. |
February 4, 2021 |
Polymeric Microneedles and Rapid Additive Manufacturing of the
Same
Abstract
The invention generally relates to microneedle devices, methods
of making same, pharmaceutical compositions comprising same, and
methods of treating a disease comprising administering same.
Specifically, the disclosed microneedle devices comprise a
plurality of biocompatible microneedles having one or more of: (i)
a curved, discontinuous, undercut, and/or perforated sidewall; (ii)
a sidewall comprising a breakable support; and (iii) a
cross-section that is non-circular and non-polygonal. The
microneedles may also be tiered. Alternatively, the microneedles
may be tiered. This abstract is intended as a scanning tool for
purposes of searching in the particular art and is not intended to
be limiting of the present invention.
Inventors: |
Desimone; Joseph M.; (Monte
Sereno, CA) ; Robbins; Gregory R.; (Redwood City,
CA) ; Johnson; Ashley R.; (Coppell, TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The University of North Carolina at Chapel Hill |
Chapel Hill |
NC |
US |
|
|
Family ID: |
1000005152404 |
Appl. No.: |
17/009569 |
Filed: |
September 1, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15557003 |
Sep 8, 2017 |
10792857 |
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PCT/US2016/022231 |
Mar 12, 2016 |
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17009569 |
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62132990 |
Mar 13, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61M 2037/0053 20130101;
A61M 2037/0046 20130101; A61B 5/150022 20130101; B29C 64/135
20170801; B29C 64/129 20170801; A61M 2037/0023 20130101; A61B
5/150282 20130101; A61M 37/0015 20130101; B29C 64/124 20170801;
A61B 5/150984 20130101 |
International
Class: |
B29C 64/124 20060101
B29C064/124; A61B 5/15 20060101 A61B005/15; B29C 64/129 20060101
B29C064/129; B29C 64/135 20060101 B29C064/135; A61M 37/00 20060101
A61M037/00 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with government support under Grant
No. HDTRA 1-13-1-0045 awarded by the Department of Defense. The
government has certain rights in the invention.
Claims
1. A microneedle device comprising: (a) a backing; and (b) a
plurality of biocompatible microneedles projecting from the
backing, and wherein the microneedles comprise one or more of: (i)
a curved, discontinuous, undercut, and/or perforated sidewall; (ii)
a sidewall comprising a breakable support; and (iii) a
cross-section that is non-circular and non-polygonal, and/or
wherein the microneedles are tiered, and wherein the microneedles
have a cross-sectional width that varies in both dimensions along
at least a portion of their length.
2. The device of claim 1, wherein the microneedles have a tip
diameter of less than about 10 micrometers.
3. The device of claim 1, wherein the backing and the microneedles
comprise the same material.
4. The device of claim 1, wherein the backing and the microneedles
comprise different materials.
5. The device of claim 1, wherein the microneedles comprise a
curved, discontinuous, or undercut sidewall.
6. The device of claim 1, wherein the microneedles comprise a
perforated sidewall.
7. The device of claim 6, wherein the sidewall is physically
perforated.
8. The device of claim 6, wherein the sidewall is chemically
perforated.
9. The device of claim 1, wherein the sidewall comprises a
breakable support.
10. The device of claim 1, wherein the cross-section is
non-circular and non-polygonal.
11. The device of claim 1, wherein the microneedles are hollow.
12. The device of claim 1, wherein the microneedles are
biodegradable and/or bioabsorbable.
13. The device of claim 1, wherein the microneedles comprise a
biodegradable and/or bioabsorbable polymer.
14. The device of claim 1, wherein the microneedles comprise a
therapeutic agent.
15. A method of delivering a therapeutic agent to a subject, the
method comprising administering to the subject a microneedle device
comprising: (a) a backing; (b) a plurality of biocompatible
microneedles projecting from the backing, wherein the microneedles
comprise a therapeutic agent and one or more of: (i) a curved,
discontinuous, undercut, or perforated sidewall; (ii) a sidewall
comprising a breakable support; and (iii) a cross-section that is
non-circular and non-polygonal, and/or wherein the microneedles are
tiered, thereby delivering the therapeutic agent.
16. The method of claim 15, wherein the therapeutic agent comprises
a protein therapeutic, a small molecule therapeutic, a vaccine
antigen, or an antigenic fragment thereof
17. A method of making a microneedle device, the method comprising
the steps of: (a) providing a build elevator and an optically
transparent build surface, wherein the build elevator and the build
surface together define a build region there between, wherein the
build surface is permeable to a polymerization inhibitor, and
wherein the build surface is in fluid communication with a source
of the polymerization inhibitor; (b) filling the build region with
a polymerizable liquid; (c) irradiating the build region through
the build surface to produce a solid polymerized region in the
build region; (d) forming or maintaining a liquid film release
layer between the solid polymerized region and the build surface,
wherein the liquid film release layer comprises the polymerizable
liquid, and wherein the polymerization of the liquid is inhibited
by the polymerization inhibitor; and (e) advancing the build
elevator away from the build surface to create a subsequent build
region between the solid polymerized region and the build surface
while concurrently filling the subsequent build region with the
polymerizable liquid, wherein the device comprises: (f) a backing;
and (g) a plurality of biocompatible microneedles projecting from
the backing, wherein the microneedles comprise one or more of: (i)
a curved, discontinuous, undercut, or perforated sidewall; (ii) a
sidewall comprising a breakable support; and (iii) a cross-section
that is non-circular and non-polygonal, and/or wherein the
microneedles are tiered, thereby making the microneedle device.
18. The method of claim 17, wherein irradiating is via actinic
radiation.
19. The method of claim 17, wherein advancing comprises moving the
build elevator vertically away from the build surface.
20. The method of claim 17, wherein the microneedle device is
formed in less than about 30 minutes.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This Application is a continuation of U.S. patent
application Ser. No. 15/557,003, filed Sep. 8, 2017, which is a
U.S. National Phase Application of International Application No.
PCT/US2016/022231, filed Mar. 12, 2016, which claims the benefit of
U.S. Provisional Application No. 62/132,990, filed on Mar. 13,
2015, the contents of which are incorporated herein by reference in
their entireties.
BACKGROUND
[0003] Polymeric microneedles are usually fabricated in three
distinct steps: master fabrication, mold fabrication, and mold
filling. Each of these steps present unique challenges that have
hindered the commercialization of microneedle technology. In a
typical process, a metal or silicon master would be created using
traditional microfabrication techniques, such as deep reactive ion
etching (DRIE), wet etching, laser ablation, or tilted ultraviolet
photolithography. Taken as a whole, master fabrication processes
are time consuming, require expensive equipment and substantial
expertise, and limit control over the shape of the resulting
microneedle. For example, dry etching techniques used to make
microneedles vertically etch on the order of 1-5 .mu.m per minute,
producing a microneedle master at upwards of .about.1.5 hours,
depending on microneedle height. Due to extensive process
optimization required to generate a microneedle structure,
substantial lead time is also required. Following the master
fabrication, a mold is then cast in polydimethylsiloxane (PDMS) and
filled with a formulation of interest using a series of vacuum and
centrifugation steps. These time-consuming vacuum and
centrifugation steps (on the order of hours to days) limit
opportunity for cost effective scale-up of manufacturing
processes.
[0004] Due to the current processing limitations, microneedle size,
shape, sharpness, aspect ratio, and spacing are therefore dictated
by feasibility of fabrication rather than ideal design. However,
numerous studies indicate that proper optimization of microneedle
morphology and spacing is essential to successful and complete
insertion into the skin. Therefore, there remains a need for
devices and methods that overcome these deficiencies and that
effectively provide polymeric microneedles.
SUMMARY
[0005] In accordance with the purpose(s) of the invention, as
embodied and broadly described herein, the invention, in one
aspect, relates to microneedle devices, methods of making same,
pharmaceutical compositions comprising same, and methods of
treating a disease comprising administering same.
[0006] Disclosed are microneedle devices comprising: (a) a backing;
and (b) a plurality of biocompatible microneedles projecting from
the backing, and wherein the microneedles comprise one or more of:
(i) a curved, discontinuous, undercut, and/or perforated sidewall;
(ii) a sidewall comprising a breakable support; and (iii) a
cross-section that is non-circular and non-polygonal, and/or
wherein the microneedles are tiered, and wherein the microneedles
have a cross-sectional width that varies in both dimensions along
at least a portion of their length.
[0007] Also disclosed are methods of delivering a therapeutic agent
to a subject, the method comprising administering to the subject a
microneedle device comprising: (a) a backing; and (b) a plurality
of biocompatible microneedles projecting from the backing, wherein
the microneedles comprise a therapeutic agent and one or more of:
(i) a curved, discontinuous, undercut, or perforated sidewall; (ii)
a sidewall comprising a breakable support; and (iii) a
cross-section that is non-circular and non-polygonal, and/or
wherein the microneedles are tiered, thereby delivering the
therapeutic agent.
[0008] Also disclosed are methods of treating a disease in a
subject, the method comprising administering to the subject a
microneedle device comprising: (a) a backing; and (b) a plurality
of biocompatible microneedles projecting from the backing, wherein
the microneedles comprise a therapeutic agent and one or more of:
(i) a curved, discontinuous, undercut, or perforated sidewall; (ii)
a sidewall comprising a breakable support; and (iii) a
cross-section that is non-circular and non-polygonal, and/or
wherein the microneedles are tiered, thereby treating the
disease.
[0009] Also disclosed are of methods of detecting a biomarker in a
sample, the method comprising: (a) providing a microneedle device
comprising: (i) a backing; and (ii) a plurality of biocompatible
microneedles projecting from the backing, wherein the microneedles
comprise a probe for the biomarker and one or more of: (1) a
curved, discontinuous, undercut, and/or perforated sidewall; (2) a
sidewall comprising a breakable support; and (3) a cross-section
that is non-circular and non-polygonal, and/or wherein the
microneedles are tiered; (b) contacting the device with the sample;
and (c) identifying the biomarker, thereby detecting the biomarker
in the sample.
[0010] Also disclosed are methods of making a disclosed microneedle
device.
[0011] Also disclosed are methods of making a microneedle device,
the method comprising the steps of: (a) providing a build elevator
and an optically transparent build surface, wherein the build
elevator and the build surface together define a build region there
between, wherein the build surface is permeable to a polymerization
inhibitor, and wherein the build surface is in fluid communication
with a source of the polymerization inhibitor; (b) filling the
build region with a polymerizable liquid; (c) irradiating the build
region through the build surface to produce a solid polymerized
region in the build region; (d) forming or maintaining a liquid
film release layer between the solid polymerized region and the
build surface, wherein the liquid film release layer comprises the
polymerizable liquid, and wherein the polymerization of the liquid
is inhibited by the polymerization inhibitor; and (e) advancing the
build elevator away from the build surface to create a subsequent
build region between the solid polymerized region and the build
surface while concurrently filling the subsequent build region with
the polymerizable liquid, wherein the device comprises: (f) a
backing; and (g) a plurality of biocompatible microneedles
projecting from the backing, wherein the microneedles comprise one
or more of: (i) a curved, discontinuous, undercut, or perforated
sidewall; (ii) a sidewall comprising a breakable support; and (iii)
a cross-section that is non-circular and non-polygonal, and/or
wherein the microneedles are tiered, thereby making the microneedle
device.
[0012] Also disclosed are microneedle devices comprising: a
backing; and a plurality of polymeric biocompatible microneedles
projecting from the backing. In various aspects, the microneedles
and/or backing are biocompatible. In various aspects, the
microneedles and/or backing are biodegradable and/or bioabsorbable.
In various aspects, the microneedles have a curved, discontinuous
or undercut sidewall. In various aspects, the microneedles have a
non-circular or non-polygonal (e.g., non-square) cross-section.
[0013] Also disclosed are methods of delivering a therapeutic agent
to a subject in need thereof, comprising administering to said
subject a microneedle device as taught herein comprising the
therapeutic agent.
[0014] Also disclosed are methods of treating a disease or
condition in a subject in need thereof, comprising administering to
said subject a microneedle device as taught herein comprising a
therapeutic agent for treatment thereof.
[0015] Also disclosed are methods of making a microneedle device as
taught herein, comprising the steps of: (a) providing a build
elevator and an optically transparent build surface defining a
build region there between, said build surface being permeable to a
polymerization inhibitor, and with said build surface in fluid
communication with a source of the polymerization inhibitor; (b)
filling said build region with a polymerizable liquid, said
polymerizable liquid contacting said build surface, (c) irradiating
said build region through said build surface to produce a solid
polymerized region in said build region, while forming or
maintaining a liquid film release layer comprised of said
polymerizable liquid formed between said solid polymerized region
and said build surface, wherein the polymerization of which liquid
film is inhibited by said polymerization inhibitor; and (d)
advancing said build elevator with said polymerized region adhered
thereto away from said build surface to create a subsequent build
region between said polymerized region and said build surface while
concurrently filling said subsequent build region with
polymerizable liquid as in step (b), to thereby form the
microneedle device.
[0016] While aspects of the present invention can be described and
claimed in a particular statutory class, such as the system
statutory class, this is for convenience only and one of skill in
the art will understand that each aspect of the present invention
can be described and claimed in any statutory class. Unless
otherwise expressly stated, it is in no way intended that any
method or aspect set forth herein be construed as requiring that
its steps be performed in a specific order. Accordingly, where a
method claim does not specifically state in the claims or
descriptions that the steps are to be limited to a specific order,
it is no way intended that an order be inferred, in any respect.
This holds for any possible non-express basis for interpretation,
including matters of logic with respect to arrangement of steps or
operational flow, plain meaning derived from grammatical
organization or punctuation, or the number or type of aspects
described in the specification.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] The accompanying figures, which are incorporated in and
constitute a part of this specification, illustrate several aspects
and together with the description serve to explain the principles
of the invention.
[0018] FIG. 1 shows representative diagrams of microneedles
engineered with breakable supports. Specifically, panel (A) shows a
representative depiction of a microneedle patch with arrows
representing individual needles. Panels (B)-(E) show representative
depictions of individual microneedles with breakable supports at
the base (panel (B)), middle (panel (C)), top (panel (D)), and
undercut (panel (E)) of the microneedle. Panels (F)-(I) show a
representative depiction of microneedles with separation at the
base (panel (F)), middle (panel (G)), top (panel (H)), and undercut
(panel (I)) of the microneedle after insertion in the skin followed
by application of torsion or other physical force.
[0019] FIG. 2 shows representative diagrams of microneedles
engineered with physical perforations. Specifically, panel (A)
shows a representative depiction of a microneedle patch with arrows
representing individual needles. Panels (B)-(D) show representative
depictions of individual microneedles with horizontal (panel (B)),
diagonal (panel (C)), or multi-directional (panel (D)) breakable
perforations. Panels (E)-(G) show representative depictions of
microneedles with separation horizontal (panel (E)), diagonal
(panel (F)), or multi-directional (panel (G)) after insertion in
the skin and application of torsion or other physical force.
[0020] FIG. 3 shows representative diagrams of breakable
microneedles engineered with chemical perforations. Specifically,
panel (A) shows a representative depiction of a microneedle patch
with arrows representing individual needles. Panels (B)-(D) show
representative depictions of individual microneedles with
horizontal (panel (B)), diagonal (panel (C)), or multi-directional
(panel (D)) chemical perforations. Panels (E)-(G) show
representative depictions of microneedles with separation
horizontal (panel (E)), diagonal (panel (F)), or multi-directional
(panel (G)) following exposure to stimuli that trigger disruption
of the chemical perforation.
[0021] FIG. 4A and FIG. 4B show representative schematics of a
traditional microneedle array and theoretical force of insertion
(4A) and a tiered microneedle array and theoretical force of
insertion (4B).
[0022] FIG. 5A-D shows representative images of CLIP microneedles
with square pyramidal (5A), curved (5B and 5C), and discontinuous
(5D) sidewalls.
[0023] FIG. 6A shows a representative image of a microneedle array
comprising microneedles having complex X-Y cross sections. FIG. 6B
shows representative images of exemplary non-circular and
non-polygonal X-Y cross-sections.
[0024] FIG. 7A shows a representative schematic of a Continuous
Liquid Interface Printer (CLIP). FIG. 7B and FIG. 7C show a
representative CLIP7 (7B) and CLIP Mini (7C).
[0025] FIG. 8A-C show representative fabricated microneedle
compositions. Specifically, polyacrylic acid microneedles (8A),
polyethylene glycol microneedles (8B), and polycaprolactone
microneedles (8C) measuring 1000 .mu.m in height and 333 .mu.m in
width are shown. Scale bars measure 500 .mu.m.
[0026] FIG. 9 shows a representative schematic of a mid-production
resin exchange.
[0027] FIG. 10A-D show representative examples of multi-component
microneedles with layers along the z-axis in which the needle tip
(10A), patch backing (10B), needle tip and patch backing (10C), and
multiple layers of the needle (10D) comprise different
compositions.
[0028] FIG. 11A-C show representative images illustrating that CLIP
enables fast print speeds and layerless part construction.
[0029] FIG. 12 shows a representative diagram indicating that CLIP
removes sequential steps from traditional stereolithography
(SL).
[0030] FIG. 13A and FIG. 13B show representative images measuring
dot thickness to quantify dead zone and cure thickness.
Specifically, FIG. 13A shows a representative schematic of a
differential dead zone thickness measurement. FIG. 13B shows a
representative image of cured thickness as a function of photon
flux and exposure time. Each exposed dot has a diameter of 3
mm.
[0031] FIG. 14 shows a representative graph illustrating that the
dead zone is created by oxygen permeation through the window.
[0032] FIG. 15A-D show representative images illustrating that a
trade-off exists between print speed and print resolution.
[0033] FIG. 16 shows a representative diagram illustrating that
maximum print speed is identical for resins with different
photoinitiator concentration.
[0034] FIG. 17 shows a representative schematic illustrating that
free radicals either inhibit oxygen or initiate polymerization.
[0035] FIG. 18A-C show representative images illustrating that
parts fabricated using CLIP can range in size from micro-paddles
with 50 .mu.M diameter stems (18A, printed at 25 mm/hr), a 10 cm
tall Eiffel Tower model (18B, printed at 100 mm/hr ; features
<1mm in size are obtained as shown in the inset), and a shoe
cleat over 20 cm long (18C, printed at 100 mm/hr).
[0036] FIG. 19A-D show representative images of CLIP microneedles
with a variety of dimensions. Specifically, FIG. 19A-C show
representative images of TMPTA microneedles with 1000 .mu.m (19A),
700 .mu.m (19B), and 400 .mu.m (19C) nominal heights height. Scale
bars are 500 .mu.m. FIG. 19D shows a representative 1800X view of a
microneedle tip with a tip radius less than 5 .mu.m. Scale bar is 5
.mu.m.
[0037] FIG. 20A-D show representative images of skin penetration of
CLIP microneedle arrays. Specifically, FIG. 20A-C show
representative images illustrating that microneedle arrays made of
TMPTA (20A), PAA (20B), and PEG (20C) on murine skin can be
visualized using a green tissue marking dye. FIG. 20D show a
representative image illustrating that no insertion sites are
visualized on a piece of control skin to which no microneedles were
applied.
[0038] FIG. 21A and FIG. 21B show representative images
illustrating that feasibility of using a microneedle patch for
insulin delivery. Specifically, FIG. 21A shows the representative
dimensions of an individual needle used for insulin loading
calculations. FIG. 21B shows the representative size of a patch
required to dose 4U of insulin.
[0039] FIG. 22A-C show representative dimensions and images CLIP
fabricated poly(acrylic acid) (PAA) cylinders. Specifically, FIG.
22A shows a representative diagram of the cylinder dimensions, FIG.
22B shows a representative image of CLIP vehicle control and
OVA-647 loaded PAA cylinders, and FIG. 22C shows a representative
florescence microscopy image of OVA-647 signal in vehicle control
and OVA-647 loaded PAA cylinders.
[0040] FIG. 23 shows a representative chart illustrating OVA-647
release from CLIP fabricated PAA cylinders.
[0041] FIG. 24 shows a representative chart illustrating R-848
bio-activity following release from CLIP fabricated PAA
cylinders.
[0042] FIG. 25 shows a representative chart illustrating human
insulin release from CLIP fabricated PAA cylinders.
[0043] FIG. 26A and FIG. 26B show a representative image (26A) and
corresponding data (26B) illustrating FITC-BSA incorporation and
release from CLIP PEG microneedles.
[0044] FIG. 27 shows a representative image of tip-loaded
microneedles produced using the mid-production resin exchange
method.
[0045] FIG. 28A-C show representative images of undercut CLIP
microneedles. Specifically, TMPTA arrowhead microneedles produced
using CLIP (28A), the dimensions of the arrowhead microneedles in
28A (28B), and other examples of undercut structures (28C).
[0046] FIG. 29 shows a representative image of a TMPTA tiered
microneedle array.
[0047] FIG. 30 shows a representative image of illustrating that
higher drug loading may result in higher insertion forces.
[0048] FIG. 31A and FIG. 31B show representative images
illustrating Tri-material microneedles with dissolvable (left) and
non-dissolvable (right) perforations made using CLIP technology
prior to exposure to water (31A) and following submersion in water
for 12 minutes (31B).
[0049] FIG. 32 shows representative images of microneedles with
dissolving (top) and non-dissolving (bottom) tips containing
rhodamine prior to skin application (left) and following 5 minute
application to porcine skin (middle). Rhodamine fluorescence in
porcine skin imaged following microneedle patch application is also
shown (right).
[0050] Additional advantages of the invention will be set forth in
part in the description which follows, and in part will be obvious
from the description, or can be learned by practice of the
invention. The advantages of the invention will be realized and
attained by means of the elements and combinations particularly
pointed out in the appended claims. It is to be understood that
both the foregoing general description and the following detailed
description are exemplary and explanatory only and are not
restrictive of the invention, as claimed.
DETAILED DESCRIPTION
[0051] The present invention can be understood more readily by
reference to the following detailed description of the invention
and the Examples included therein.
[0052] Before the present compounds, compositions, articles,
systems, devices, and/or methods are disclosed and described, it is
to be understood that they are not limited to specific synthetic
methods unless otherwise specified, or to particular reagents
unless otherwise specified, as such may, of course, vary. It is
also to be understood that the terminology used herein is for the
purpose of describing particular aspects only and is not intended
to be limiting. Although any methods and materials similar or
equivalent to those described herein can be used in the practice or
testing of the present invention, example methods and materials are
now described.
[0053] Throughout this application, various publications are
referenced. The disclosures of these publications in their
entireties are hereby incorporated by reference into this
application in order to more fully describe the state of the art to
which this pertains. The references disclosed are also individually
and specifically incorporated by reference herein for the material
contained in them that is discussed in the sentence in which the
reference is relied upon. Further, the dates of publication
provided herein may be different from the actual publication dates,
which can require independent confirmation.
A. DEFINITIONS
[0054] The present invention now will be described more fully
hereinafter with reference to the accompanying drawings, in which
illustrative aspects of the invention are shown. In the drawings,
the relative sizes of regions or features may be exaggerated for
clarity. This invention may, however, be embodied in many different
forms and should not be construed as limited to the aspects set
forth herein; rather, these aspects are provided so that this
disclosure will be thorough and complete, and will fully convey the
scope of the invention to those skilled in the art.
[0055] In addition, spatially relative terms, such as "under,"
"below," "lower," "over," "upper," and the like, may be used herein
for ease of description to describe one element or feature's
relationship to another element(s) or feature(s) as illustrated in
the figures. It will be understood that the spatially relative
terms are intended to encompass different orientations of the
device in use or operation in addition to the orientation depicted
in the figures. For example, if the device in the figures is
inverted, elements described as "under" or "beneath" other elements
or features would then be oriented "over" the other elements or
features. Thus, the exemplary term "under" can encompass both an
orientation of over and under. The device may be otherwise oriented
(rotated 90 degrees or at other orientations) and the spatially
relative descriptors used herein interpreted accordingly.
[0056] It will be understood that when an element is referred to as
being "coupled" or "connected" to another element, it can be
directly coupled or connected to the other element or intervening
elements may also be present. In contrast, when an element is
referred to as being "directly coupled" or "directly connected" to
another element, there are no intervening elements present. Like
numbers refer to like elements throughout. As used herein the term
"and/or" includes any and all combinations of one or more of the
associated listed items.
[0057] Well-known functions or constructions may not be described
in detail for brevity and/or clarity.
[0058] The disclosures of all patent references cited herein are
hereby incorporated by reference to the extent they are consistent
with the disclosure set forth herein. As used herein in the
description of the invention and the appended claims, the singular
forms "a," "an," and "the" are intended to include the plural forms
as well, unless the context clearly indicates otherwise.
[0059] Unless otherwise defined, all terms (including technical and
scientific terms) used herein have the same meaning as commonly
understood by one of ordinary skill in the art to which this
invention belongs. It will be further understood that terms, such
as those defined in commonly used dictionaries, should be
interpreted as having a meaning that is consistent with their
meaning in the context of the present application and relevant art
and should not be interpreted in an idealized or overly formal
sense unless expressly so defined herein. The terminology used in
the description of the invention herein is for the purpose of
describing particular aspects only and is not intended to be
limiting of the invention. All publications, patent applications,
patents and other references mentioned herein are incorporated by
reference in their entirety. In case of a conflict in terminology,
the present specification is controlling.
[0060] Also as used herein, "and/or" refers to and encompasses any
and all possible combinations of one or more of the associated
listed items, as well as the lack of combinations when interpreted
in the alternative ("or").
[0061] Unless the context indicates otherwise, it is specifically
intended that the various features of the invention described
herein can be used in any combination. Moreover, the present
invention also contemplates that In various aspects of the
invention, any feature or combination of features set forth herein
can be excluded or omitted. To illustrate, if the specification
states that a complex comprises components A, B and C, it is
specifically intended that any of A, B or C, or a combination
thereof, can be omitted and disclaimed.
[0062] The term "about," as used herein when referring to a
measurable value, such as, for example, an amount or concentration
and the like, is meant to encompass variations of .+-.20%, .+-.10%,
.+-.5%, .+-.1%, .+-.0.5%, or even .+-.0.1% of the specified amount.
A range provided herein for a measureable value may include any
other range and/or individual value therein.
[0063] As used herein, "biocompatible" refers to materials that are
not unduly reactive or harmful to a subject upon
administration.
[0064] "Biodegradable" as used herein refers to the ability of a
material to be broken down in vivo upon administration to a
subject. For example, the materials may be dissolvable in skin
tissue. See, e.g., Lee et al., "Dissolving Microneedles for
Transdermal Drug Delivery," Biomaterials 29(13):2113-2124, 2008. In
various aspects, materials may be chosen to biodegrade at a
predetermined rate, e.g., for controlled delivery of a therapeutic
agent cargo.
[0065] "Bioabsorbable" as used herein means capable of being
absorbed into living tissue.
[0066] The amount of agents that may be incorporated in the
microneedles described herein can vary from picogram levels to
milligram levels, depending on the size of microneedles and/or
encapsulation efficiency. Non-limiting examples of agents include
organic materials such as horseradish peroxidase,
phenolsulfonphthalein, nucleotides, nucleic acids (e.g.,
oligonucleotides, polynucleotides, siRNA, shRNA), aptamers,
antibodies or portions thereof (e.g., antibody-like molecules),
hormones (e.g., insulin, testosterone), growth factors, enzymes
(e.g., peroxidase, lipase, amylase, organophosphate dehydrogenase,
ligases, restriction endonucleases, ribonucleases, RNA or DNA
polymerases, glucose oxidase, lactase), cells (e.g., red blood
cells, stem cells), bacteria or viruses, other proteins or
peptides, small molecules (e.g., drugs, dyes, amino acids,
vitamins, antioxidants), lipids, carbohydrates, chromophores, light
emitting organic compounds (such as luciferin, carotenes) and light
emitting inorganic compounds (e.g., chemical dyes and/or contrast
enhancing agents such as indocyanine green), immunogenic substances
such as vaccines, antibiotics, antifungal agents, antiviral agents,
therapeutic agents, diagnostic agents or pro-drugs, analogs or
combinations of any of the foregoing.
[0067] Examples of immunogenic vaccine substances that can be
included in the microneedles described herein include, but are not
limited to, those in BIOTHRAX.RTM. (anthrax vaccine adsorbed,
Emergent Biosolutions, Rockville, Md.); TICE.RTM. BCG Live
(Bacillus Calmette-Guerin for intravesical use, Organon Tekina
Corp. LLC, Durham, N.C.); MYCOBAX .RTM. BCG Live (Sanofi Pasteur
Inc); DAPTACEL.RTM. (diphtheria and tetanus toxoids and acellular
pertussis [DTaP] vaccine adsorbed, Sanofi Pasteur Inc.);
INFANRIX.RTM. (DTaP vaccine adsorbed, GlaxoSmithKline);
TRIPEDIA.RTM. (DTaP vaccine, Sanofi Pasteur); TRIHIBIT.RTM.
(DTaP/Hib#, sanofi pasteur); KINRIX.phi. (diphtheria and tetanus
toxoids, acellular pertussis adsorbed and inactivated poliovirus
vaccine, GlaxoSmithKline); PEDIARIX.RTM. (DTaP-HepB-IPV,
GlaxoSmithKline); PENTACEL.RTM. (diphtheria and tetanus toxoids and
acellular pertussis adsorbed, inactivated poliovirus and
Haemophilus b conjugate [tetanus toxoid conjugate] vaccine, sanofi
pasteur); Diphtheria and Tetanus Toxoids, adsorbed (for pediatric
use, Sanofi Pasteur); DECAVAC.RTM. (diphtheria and tetanus toxoids
adsorbed, for adult use, Sanofi Pasteur); ACTHIB.RTM. (Haemophilus
b tetanus toxoid conjugate vaccine, Sanofi Pasteur); PEDVAXHIB.RTM.
(Hib vaccine, Merck); Hiberix (Haemophilus b tetanus toxoid
conjugate vaccine, booster dose, GlaxoSmithKline); COMVAX.RTM.
(Hepatitis B-Hib vaccine, Merck); HAVRIX.RTM. (Hepatitis A vaccine,
pediatric, GlaxoSmithKline); VAQTA.RTM. (Hepatitis A vaccine,
pediatric, Merck); ENGERIX-B.RTM. (Hep B, pediatric, adolescent,
GlaxoSmithKline); RECOMBIVAX HB.RTM. (hepatitis B vaccine, Merck);
TWINRIX.RTM. (HepA/HepB vaccine, 18 years and up, GlaxoSmithKline);
CERVARIX.RTM. (human papillomavirus bivalent [types 16 and 18]
vaccine, recombinant, GlaxoSmithKline); GARDASIL.RTM. (human
papillomavirus bivalent [types 6, 11, 16 and 18] vaccine,
recombinant, Merck); AFLURIA.RTM. (Influenza vaccine, 18 years and
up, CSL); AGRIFLU.TM. (influenza virus vaccine for intramuscular
injection, Novartis Vaccines); FLUARIX.RTM. (Influenza vaccine, 18
years and up, GaxoSmithKline); FLULAVAL.RTM. (Influenza vaccine, 18
years and up, GlaxoSmithKline); FLUVIRIN.RTM. (Influenza vaccine, 4
years and up, Novartis Vaccine); FLUZONE.RTM. (Influenza vaccine, 6
months and up, Sanofi Pasteur); FLUMIST.RTM. (Influenza vaccine, 2
years and up, Medlmmune); IPOL.RTM. (e-IPV polio vaccine, sanofi
Pasteur); JE VAX.RTM. (Japanese encephalitis virus vaccine
inactivated, BIKEN, Japan); IXIARO.RTM. (Japanese encephalitis
virus vaccine inactivated, Novarits); MENACTRA.RTM. (Meningococcal
[Groups A, C, Y and W-135] and diphtheria vaccine, Sanofi Pasteur);
MENOMUNE.RTM.-A/C/Y/W-135 (Meningococcal polysaccharide vaccine,
sanofi pasteur); MMRII.RTM. (MMR vaccine, Merck); MENVEO.RTM.
(Meningococcal [Groups A, C, Y and W-135] oligosaccharide
diphtheria CRM 197 conjugate vaccine, Novartis Vaccines);
PROQUAD.RTM. (MMR and varicella vaccine, Merck); PNEUMOVAX 23.RTM.
(pneumococcal polysaccharide vaccine, Merck); PREVNAR.RTM.
(pneumococcal vaccine, 7-valent, Wyeth/Lederle); PREVNAR-13C)
(pneumococcal vaccine, 13-valent, Wyeth/Lederle); POLIO VAX.TM.
(poliovirus inactivated, sanofi pasteur); IMOVAX.RTM. (Rabies
vaccine, Sanofi Pasteur); RABAVERT.TM. (Rabies vaccine, Chiron);
ROTATEQ.RTM. (Rotavirus vaccine, live, oral pentavalent, Merck);
ROTARIX.RTM. (Rotavirus, live, oral vaccine, GlaxoSmithKline);
DECAVAC.TM. (tetanus and diphtheria toxoids vaccine, sanofi
pasteur); Td (generic) (tetanus and diphtheria toxoids, adsorbed,
Massachusetts Biol. Labs); TYPHIMVI.RTM. (typhoid Vi polysaccharide
vaccine, Sanofi Pasteur); ADACEL.RTM. (tetanus toxoid, reduced
diphtheria toxoid and acellular pertussis, sanofi pasteur);
BOOSTRIX.RTM. (tetanus toxoid, reduced diphtheria toxoid and
acellular pertussis, GlaxoSmithKline); VIVOTIF.RTM. (typhoid
vaccine live oral Ty21a, Berna Biotech); ACAM2000.TM. (Smallpox
(vaccinia) vaccine, live, Acambis, Inc.); DRYVAX.RTM. (Smallpox
(vaccinia) vaccine); VARIVAX.RTM. (varicella [live] vaccine,
Merck); YF-VAX.RTM. (Yellow fever vaccine, Sanofi Pasteur);
ZOSTAVAX.RTM. (Varicella zoster, Merck); or combinations thereof.
Any vaccine products listed in database of Center for Disease
Control and Prevention (CDC) can also be included in the
compositions described herein.
[0068] As used herein, "small molecule" refers to natural or
synthetic molecules including, but not limited to, peptides,
peptidomimetics, amino acids, amino acid analogs, polynucleotides,
polynucleotide analogs, aptamers, nucleotides, nucleotide analogs,
organic or inorganic compounds (i.e., including heteroorganic and
organometallic compounds) having a molecular weight less than about
10,000 grams per mole, organic or inorganic compounds having a
molecular weight less than about 5,000 grams per mole, organic or
inorganic compounds having a molecular weight less than about 1,000
grams per mole, organic or inorganic compounds having a molecular
weight less than about 500 grams per mole, and salts, esters, and
other pharmaceutically acceptable forms of such compounds.
[0069] The term "antibiotic" is used herein to describe a compound
that acts as an antimicrobial, bacteriostatic, or bactericidal
agent. Example antibiotics include, but are not limited to,
penicillins, cephalosporins, penems, carbapenems, monobactams,
aminoglycosides, sulfonamides, macrolides, tetracyclins,
lincosides, quinolones, chloramphenicol, vancomycin, metronidazole,
rifampin, isoniazid, spectinomycin, trimethoprim, and
sulfamethoxazole.
[0070] The term "therapeutic agent" is art-recognized and refers to
any chemical moiety that is a biologically, physiologically, or
pharmacologically active substance that acts locally or
systemically in a subject. Examples of therapeutic agents, also
referred to as "drugs," are described in well-known literature
references such as the Merck Index, the Physicians Desk Reference,
and The Pharmacological Basis of Therapeutics, and they include,
without limitation, medicaments; vitamins; mineral supplements;
substances used for the treatment, prevention, diagnosis, cure or
mitigation of a disease or illness; substances which affect the
structure or function of the body; or pro-drugs, which become
biologically active or more active after they have been placed in a
physiological environment. Various forms of a therapeutic agent may
be used which are capable of being released from the subject
composition into adjacent tissues or fluids upon administration to
a subject. Examples include steroids and esters of steroids (e.g.,
estrogen, progesterone, testosterone, androsterone, cholesterol,
norethindrone, digoxigenin, cholic acid, deoxycholic acid, and
chenodeoxycholic acid), boron-containing compounds (e.g.,
carborane), chemotherapeutic nucleotides, drugs (e.g., antibiotics,
antivirals, antifungals), enediynes (e.g., calicheamicins,
esperamicins, dynemicin, neocarzino statin chromophore, and
kedarcidin chromophore), heavy metal complexes (e.g., cisplatin),
hormone antagonists (e.g., tamoxifen), non-specific (non-antibody)
proteins (e.g., sugar oligomers), oligonucleotides (e.g., antisense
oligonucleotides that bind to a target nucleic acid sequence (e.g.,
mRNA sequence)), peptides, proteins, antibodies, photodynamic
agents (e.g., rhodamine 123), radionuclides (e.g., I-131, Re-186,
Re-188, Y-90, Bi-212, At-211, Sr-89, Ho-166, Sm-153, Cu-67 and
Cu-64), toxins (e.g., ricin), and transcription-based
pharmaceuticals.
[0071] In various aspects, the therapeutic agent can include a pain
medication. Examples of pain medications include, but are not
limited to, acetaminophen, non-steroidal antiinflammatory
medications (NSAIDs), corticosteroids; narcotics; anti-convulsants;
local anesthetics, and any combinations thereof. NSAIDs that can be
included In various aspects of the microneedles provided herein
include, but not limited to, ibuprofen, naproxin, aspirin,
fenoprofen, flurbiprofen, ketoprofen, oxaprozin, diclofenac sodium,
etodolac, indomethacin, ketorolac, sulindac, tolmetin,
meclofenamate, mefenamic acid, nabumetone, piroxicam and COX-2
inhibitors. In various aspects, the pain medications can include
acetaminophen combinations (e.g., acetaminophen with a narcotic)
such as acetaminophen with codeine; acetaminophen with hydrocodone;
and acetaminophen with oxycodone.
[0072] In various aspects of the device, the substrate can be
formed from any flexible material. In such aspects, the substrate
can be sufficiently flexible to conform to a surface upon contact
with the surface, e.g., a tissue or an organ surface, while
allowing the microneedles to penetrate the tissue to the desired
depth. In one aspect, the flexible substrate comprises a silk
fibroin film integrated with silk fibroin microneedles. In
alternative aspects, the substrate can be any rigid material.
[0073] In various aspects the microneedles are provided in the form
of a patch which comprises a backing and a plurality of
microneedles projecting from the backing. The backing can be made
of the same material or a different material, and can have a
substantially flat surface, a curved surface, a wavy surface or any
combination thereof. In various aspects, the surface is configured
to have a curvature profile similar to that of a target surface to
be penetrated.
[0074] The backing can be of any shape and/or any dimension
determined from, for example, design of the microneedle device,
area/shape of a target site to be treated, and/or size of
microneedle applicators. In various aspects, the shape and
dimension of the backing can be configured to fit any applicator
that currently uses hypodermic needles as the barrier penetration
method (e.g., syringes), any microinjection equipment, any
microneedle holders, any microneedle administration or applicator
devices, any microneedle array applicator devices, and/or
microneedle array cartridge systems. Non-limiting examples of the
microneedle or microneedle array injectors or applicators include
the ones described in U.S. Patent Application Nos.: US
2008/0183144; US 2003/0208167; US 2010/0256597; and U.S. Pat. Nos.
6,743,211; and 7,842,008. See also US 2013/0338632 to Kaplan et
al.
[0075] In various aspects, microneedles as taught herein may be
hollow and/or porous. See, e.g., Burton et al., "Rapid Intradermal
Delivery of Liquid Formulations Using a Hollow Microstructured
Array," Pharm. Res. 28:31-40, 2011.
B. MICRONEEDLE (MN) DEVICES
[0076] In one aspect, disclosed are microneedle devices comprising:
(a) a backing; and (b) a plurality of biocompatible microneedles
projecting from the backing, and wherein the microneedles comprise
one or more of: (i) a curved, discontinuous, undercut, and/or
perforated sidewall; (ii) a sidewall comprising a breakable
support; and (iii) a cross-section that is non-circular and
non-polygonal, and/or wherein the microneedles are tiered, and
wherein the microneedles have a cross-sectional width that varies
in both dimensions along at least a portion of their length.
[0077] In one aspect, disclosed are microneedle devices comprising:
(a) a backing; and (b) a plurality of biocompatible microneedles
projecting from the backing, wherein said biocompatible
microneedles are biodegradable and/or bioabsorbable, and/or wherein
said microneedles have a curved, discontinuous or undercut sidewall
and/or have a non-circular or non-polygonal (e.g., non-square)
cross-section.
[0078] In a further aspect, the microneedles have an average
diameter of from 5, 10, 25, 50 or 100, to 250, 500, 750 or 1,000
micrometers, and/or an average length of from 5, 10, 25, 50 or 100,
to 250, 500, 750 or 1,000 micrometers, and/or an average distance
from one another of from 5, 10, 25, 50 or 100, to 250, 500, 750 or
1,000 micrometers.
[0079] In a further aspect, the microneedles have an average
diameter of from 5 to 1,000 micrometers, and/or an average length
of from 5 to 1,500 micrometers, and/or an average distance from one
another of from 5 to 1,000 micrometers.
[0080] In a further aspect, the microneedles have an average
diameter of from about 5 micrometers to about 1,000 micrometers
and/or an average length of from about 5 micrometers to about 1,500
micrometers, and/or an average distance of from about 5 micrometers
to about 1,000 micrometers from one another. In a still further
aspect, the microneedles have an average diameter of from about 5
micrometers to about 1,000 micrometers and an average length of
from about 5 micrometers to about 1,500 micrometers, and an average
distance of from about 5 micrometers to about 1,000 micrometers
from one another. In yet a further aspect, the microneedles have an
average diameter of from about 5 micrometers to about 1,000
micrometers and an average length of from about 5 micrometers to
about 1,500 micrometers, or an average distance of from about 5
micrometers to about 1,000 micrometers from one another. In an even
further aspect, the microneedles have an average diameter of from
about 5 micrometers to about 1,000 micrometers or an average length
of from about 5 micrometers to about 1,500 micrometers, and an
average distance of from about 5 micrometers to about 1,000
micrometers from one another. In a still further aspect, the
microneedles have an average diameter of from about 5 micrometers
to about 1,000 micrometers or an average length of from about 5
micrometers to about 1,500 micrometers, or an average distance of
from about 5 micrometers to about 1,000 micrometers from one
another.
[0081] In a further aspect, the microneedles have an average
diameter of from about 5 micrometers to about 1,000 micrometers. In
a still further aspect, the microneedles have an average diameter
of from about 5 micrometers to about 750 micrometers. In yet a
further aspect, the microneedles have an average diameter of from
about 5 micrometers to about 500 micrometers. In an even further
aspect, the microneedles have an average diameter of from about 5
micrometers to about 250 micrometers. In a still further aspect,
the microneedles have an average diameter of from about 5
micrometers to about 100 micrometers. In yet a further aspect, the
microneedles have an average diameter of from about 5 micrometers
to about 50 micrometers. In an even further aspect, the
microneedles have an average diameter of from about 5 micrometers
to about 25 micrometers. In a still further aspect, the
microneedles have an average diameter of from about 5 micrometers
to about 10 micrometers. In yet a further aspect, the microneedles
have an average diameter of from about 10 micrometers to about
1,000 micrometers. In an even further aspect, the microneedles have
an average diameter of from about 25 micrometers to about 1,000
micrometers. In a still further aspect, the microneedles have an
average diameter of from about 50 micrometers to about 1,000
micrometers. In yet a further aspect, the microneedles have an
average diameter of from about 100 micrometers to about 1,000
micrometers. In an even further aspect, the microneedles have an
average diameter of from about 250 micrometers to about 1,000
micrometers. In a still further aspect, the microneedles have an
average diameter of from about 500 micrometers to about 1,000
micrometers. In yet a further aspect, the microneedles have an
average diameter of from about 750 micrometers to about 1,000
micrometers.
[0082] In a further aspect, the microneedles have an average length
of from about 5 micrometers to about 1,000 micrometers. In a still
further aspect, the microneedles have an average length of from
about 5 micrometers to about 750 micrometers. In yet a further
aspect, the microneedles have an average length of from about 5
micrometers to about 500 micrometers. In an even further aspect,
the microneedles have an average length of from about 5 micrometers
to about 250 micrometers. In a still further aspect, the
microneedles have an average length of from about 5 micrometers to
about 100 micrometers. In yet a further aspect, the microneedles
have an average length of from about 5 micrometers to about 50
micrometers. In an even further aspect, the microneedles have an
average length of from about 5 micrometers to about 25 micrometers.
In a still further aspect, the microneedles have an average length
of from about 5 micrometers to about 10 micrometers. In yet a
further aspect, the microneedles have an average length of from
about 10 micrometers to about 1,000 micrometers. In an even further
aspect, the microneedles have an average length of from about 25
micrometers to about 1,000 micrometers. In a still further aspect,
the microneedles have an average length of from about 50
micrometers to about 1,000 micrometers. In yet a further aspect,
the microneedles have an average length of from about 100
micrometers to about 1,000 micrometers. In an even further aspect,
the microneedles have an average length of from about 250
micrometers to about 1,000 micrometers. In a still further aspect,
the microneedles have an average length of from about 500
micrometers to about 1,000 micrometers. In yet a further aspect,
the microneedles have an average length of from about 750
micrometers to about 1,000 micrometers.
[0083] In a further aspect, the microneedles have an average
distance of from about 5 micrometers to about 1,000 micrometers
from one another. In a still further aspect, the microneedles have
an average distance of from about 5 micrometers to about 750
micrometers. In yet a further aspect, the microneedles have an
average distance of from about 5 micrometers to about 500
micrometers. In an even further aspect, the microneedles have an
average distance of from about 5 micrometers to about 250
micrometers. In a still further aspect, the microneedles have an
average distance of from about 5 micrometers to about 100
micrometers. In yet a further aspect, the microneedles have an
average distance of from about 5 micrometers to about 50
micrometers. In an even further aspect, the microneedles have an
average distance of from about 5 micrometers to about 25
micrometers. In a still further aspect, the microneedles have an
average distance of from about 5 micrometers to about 10
micrometers. In yet a further aspect, the microneedles have an
average distance of from about 10 micrometers to about 1,000
micrometers. In an even further aspect, the microneedles have an
average distance of from about 25 micrometers to about 1,000
micrometers. In a still further aspect, the microneedles have an
average distance of from about 50 micrometers to about 1,000
micrometers. In yet a further aspect, the microneedles have an
average distance of from about 100 micrometers to about 1,000
micrometers. In an even further aspect, the microneedles have an
average distance of from about 250 micrometers to about 1,000
micrometers. In a still further aspect, the microneedles have an
average distance of from about 500 micrometers to about 1,000
micrometers. In yet a further aspect, the microneedles have an
average distance of from about 750 micrometers to about 1,000
micrometers.
[0084] In a further aspect, the microneedles have a tip diameter of
less than 20, 15, 10, 8, 5, or 3 micrometers. In a still further
aspect, the microneedles have a tip diameter of less than 20, 15,
10, 8, or 5 micrometers. In yet a further aspect, the microneedles
have a tip diameter of less than 20, 15, 10, or 8 micrometers. In
an even further aspect, the microneedles have a tip diameter of
less than 20, 15, or 10 micrometers. In a still further aspect, the
microneedles have a tip diameter of less than 20 or 15 micrometers.
In yet a further aspect, the microneedles have a tip diameter of
less than 15, 10, 8, 5, or 3 micrometers. In an even further
aspect, the microneedles have a tip diameter of less than 10, 8, 5,
or 3 micrometers. In a still further aspect, the microneedles have
a tip diameter of less than 8, 5, or 3 micrometers. In yet a
further aspect, the microneedles have a tip diameter of less than 5
or 3 micrometers.
[0085] In a further aspect, the microneedles have a tip diameter of
less than about 20 micrometers. In a still further aspect, the
microneedles have a tip diameter of less than about 15 micrometers.
In yet a further aspect, the microneedles have a tip diameter of
less than about 10 micrometers. In an even further aspect, the
microneedles have a tip diameter of less than about 8 micrometers.
In a still further aspect, the microneedles have a tip diameter of
less than about 5 micrometers. In yet a further aspect, the
microneedles have a tip diameter of less than about 3
micrometers.
[0086] In a further aspect, the backing and the microneedles
comprise the same material. In a still further aspect, the backing
and the microneedles comprise different materials.
[0087] In a further aspect, the microneedles comprise a polymer. In
a still further aspect, the microneedles comprise more than one
polymer. In a still further aspect, the microneedles are
metal-free. In yet a further aspect, the microneedles comprise less
than about 0.01 wt % metal. In an even further aspect, the
microneedles comprise less than about 0.1 wt % metal. In a still
further aspect, the microneedles comprise less than about 1 wt %
metal. In yet a further aspect, the microneedles comprise less than
about 5 wt % metal. In an even further aspect, the microneedles
comprise less than about 10 wt % metal. In a still further aspect,
the microneedles comprise less than about 25 wt % metal. In yet a
further aspect, the microneedles comprise less than about 50 wt %
metal. In an even further aspect, the microneedles comprise less
than about 75 wt % metal. In a still further aspect, the
microneedles comprise less than about 90 wt % metal. In yet a
further aspect, the microneedles comprise less than about 95 wt %
metal. In an even further aspect, the microneedles comprise less
than about 99 wt % metal. In a still further aspect, the
microneedles comprise metal.
[0088] In a further aspect, the microneedles have a cross-sectional
width that varies in both dimensions along at least a portion of
their length. In a still further aspect, the microneedles have a
cross-sectional width that varies in both dimensions along their
entire length. Thus, in various aspects, the length of the
microneedles is not flat in either dimension.
[0089] In a further aspect, the microneedles were not produced via
an "in-plane" or an "out-of-plane" technique. In a still further
aspect, the microneedles were not produced via an "in-plane"
technique. In yet a further aspect, the microneedles were not
produced via an "out-of-plane" technique.
[0090] In a further aspect, the backing comprises a cross-section.
In a still further aspect, the cross-section of the microneedle has
a thickness different from the thickness of the cross-section of
the backing. In yet a further aspect, the thickness of the
cross-section of the microneedle is greater than the thickness of
the cross-section of the backing. In an even further aspect, the
thickness of the cross-section of the microneedle is less than the
thickness of the cross-section of the backing. Accordingly, in
various aspects, the microneedles were not punched out from the
backing.
[0091] In a further aspect, the shape and/or thickness of the
cross-section of the microneedles is not limited by the shape
and/or thickness of the cross-section of the backing.
[0092] In a further aspect, the backing is free of cuts. In a still
further aspect, the backing is free of holes. In yet a further
aspect, the backing is free of cuts, wherein the cuts were used to
form the microneedles. In an even further aspect, the backing is
free of holes, wherein the holes were used to form the
microneedles.
[0093] In a further aspect, the microneedles comprise a curved,
discontinuous, undercut, or perforated sidewall. In a still further
aspect, the microneedles comprise a curved, discontinuous, or
undercut sidewall. In yet a further aspect, the microneedles
comprise a curved sidewall. In an even further aspect, the
microneedles comprise a discontinuous sidewall. In a still further
aspect, the microneedles comprise an undercut sidewall. In yet a
further aspect, the microneedles comprise a perforated
sidewall.
[0094] In a further aspect, the microneedles comprise a sidewall
comprising a breakable support.
[0095] In a further aspect, the microneedles comprise a
cross-section that is non-circular and non-polygonal.
[0096] In a further aspect, the microneedles are hollow.
[0097] In a further aspect, the microneedles are tiered.
[0098] In a further aspect, the microneedles are dissolvable. In a
still further aspect, the entire microneedle is dissolvable. In yet
a further aspect, a portion of the microneedle is dissolvable such
as, for example, the tip of the microneedle. Thus, in various
aspects, the microneedles dissolve at a rate of from about one
minute per patch to about one month per patch. In a further aspect,
the microneedles dissolve at a rate of from about one minute per
patch to about two weeks per patch. In a still further aspect, the
microneedles dissolve at a rate of from about one minute per patch
to about one week per patch. In yet a further aspect, the
microneedles dissolve at a rate of from about one minute per patch
to about 3 days per patch. In an even further aspect, the
microneedles dissolve at a rate of from about one minute per patch
to about one day per patch. In a still further aspect, the
microneedles dissolve at a rate of from about one minute per patch
to about 12 hours per patch. In yet a further aspect, the
microneedles dissolve at a rate of from about one minute per patch
to about 6 hours per patch. In an even further aspect, the
microneedles dissolve at a rate of from about one minute per patch
to about one hour per patch. In a still further aspect, the
microneedles dissolve at a rate of from about one minute per patch
to about 30 minutes per patch. In yet a further aspect, the
microneedles dissolve at a rate of from about 30 minutes per patch
to about one month per patch. In an even further aspect, the
microneedles dissolve at a rate of from about one hour per patch to
about one month per patch. In a still further aspect, the
microneedles dissolve at a rate of from about 6 hours per patch to
about one month per patch. In yet a further aspect, the
microneedles dissolve at a rate of from about 12 hours per patch to
about one month per patch. In an even further aspect, the
microneedles dissolve at a rate of from about one day per patch to
about one month per patch. In a still further aspect, the
microneedles dissolve at a rate of from about 3 days per patch to
about one month per patch. In yet a further aspect, the
microneedles dissolve at a rate of from about one week per patch to
about one month per patch. In yet a further aspect, the
microneedles dissolve at a rate of from about two weeks per patch
to about one month per patch.
[0099] In a further aspect, the microneedles dissolve at a rate of
less than about one minute per patch. In a still further aspect,
the microneedles dissolve at a rate of from about 1 second per
patch to about one minute per patch. In yet a further aspect, the
microneedles dissolve at a rate of from about 1 second per patch to
about thirty seconds per patch. In an even further aspect, the
microneedles dissolve at a rate of from about 1 second per patch to
about 10 seconds per patch. In a still further aspect, the
microneedles dissolve at a rate of from about 10 seconds per patch
to about one minute per patch. In yet a further aspect, the
microneedles dissolve at a rate of from about 30 seconds per patch
to about one minute per patch.
[0100] In a further aspect, the microneedles are biodegradable
and/or bioabsorbable. In a still further aspect, the microneedles
are biodegradable. In yet a further aspect, the microneedles are
bioabsorbable. In an even further aspect, the microneedles are
biodegradable and bioabsorbable.
[0101] In a further aspect, the microneedles comprise a
biodegradable and/or bioabsorbable polymer. In a still further
aspect, the microneedles comprise at least two biodegradable and/or
bioabsorbable polymers. In yet a further aspect, the polymer is
selected from poly(ethylene glycol), poly(caprolactone), and
polyacrylic acid, or a combination thereof.
[0102] In a further aspect, the microneedles comprise a therapeutic
agent. In a further aspect, the therapeutic agent comprises a
protein therapeutic or a small molecule therapeutic. In yet a
further aspect, the therapeutic agent is coated onto or dispersed
in said microneedles. In an even further aspect, the therapeutic
agent is coated onto the microneedles. In a still further aspect,
the therapeutic agent is dispersed in the microneedles.
[0103] In a further aspect, the therapeutic agent is released from
the microneedles. The release of the therapeutic agent may occur
upon insertion or over a period of time. For example, in various
aspects, the therapeutic agent may be released from the microneedle
over a time period of about 1 minute to about 6 months. In a
further aspect, the therapeutic agent may be released from the
microneedle over a time period of about 1 minute to about 3 months.
In a still further aspect, the therapeutic agent may be released
from the microneedle over a time period of about 1 minute to about
1 month. In yet a further aspect, the therapeutic agent may be
released from the microneedle over a time period of about 1 minute
to about 2 weeks. In an even further aspect, the therapeutic agent
may be released from the microneedle over a time period of about 1
minute to about 1 week. In a still further aspect, the therapeutic
agent may be released from the microneedle over a time period of
about 1 minute to about 3 days. In yet a further aspect, the
therapeutic agent may be released from the microneedle over a time
period of about 1 minute to about 1 day. In an even further aspect,
the therapeutic agent may be released from the microneedle over a
time period of about 1 minute to about 12 hours. In a still further
aspect, the therapeutic agent may be released from the microneedle
over a time period of about 1 minute to about 6 hours. In yet a
further aspect, the therapeutic agent may be released from the
microneedle over a time period of about 1 minute to about 1 hour.
In an even further aspect, the therapeutic agent may be released
from the microneedle over a time period of about 1 minute to about
30 minutes. In a still further aspect, the therapeutic agent may be
released from the microneedle over a time period of about 30
minutes to about 6 months. In yet a further aspect, the therapeutic
agent may be released from the microneedle over a time period of
about 1 hour to about 6 months. In an even further aspect, the
therapeutic agent may be released from the microneedle over a time
period of about 6 hours to about 6 months. In a still further
aspect, the therapeutic agent may be released from the microneedle
over a time period of about 12 hours to about 6 months. In yet a
further aspect, the therapeutic agent may be released from the
microneedle over a time period of about 1 day to about 6 months. In
an even further aspect, the therapeutic agent may be released from
the microneedle over a time period of about 3 days to about 6
months. In a still further aspect, the therapeutic agent may be
released from the microneedle over a time period of about 1 week to
about 6 months. In yet a further aspect, the therapeutic agent may
be released from the microneedle over a time period of about 2
weeks to about 6 months. In an even further aspect, the therapeutic
agent may be released from the microneedle over a time period of
about 1 month to about 6 months. In a still further aspect, the
therapeutic agent may be released from the microneedle over a time
period of about 3 months to about 6 months.
[0104] In a further aspect, the therapeutic agent may be released
from the microneedle over a time period of less than about 1
minute. In a still further aspect, the therapeutic agent may be
released from the microneedle over a time period of about 1 second
to about 1 minute. In yet a further aspect, the therapeutic agent
may be released from the microneedle over a time period of about 1
second to about 30 seconds. In an even further aspect, the
therapeutic agent may be released from the microneedle over a time
period of about 1 second to about 10 seconds. In a still further
aspect, the therapeutic agent may be released from the microneedle
over a time period of about 10 seconds to about 1 minute. In yet a
further aspect, the therapeutic agent may be released from the
microneedle over a time period of about 30 seconds to about 1
minute.
[0105] 1. Breakable Microneedles
[0106] In a further aspect, disclosed are breakable microneedles. A
microneedle may be breakable, for example, due to the shape of the
microneedle (e.g., due to the presence of holes or a thinner
structure). Alternatively, a microneedle may be breakable due to a
difference in the mechanical properties of the support, as compared
to the remainder of the microneedle.
[0107] A breakable microneedle may be broken intentionally to
remove the microneedles embedded in the skin from the patch backing
on the skin surface. Without wishing to be bound by theory, removal
of the patch backing may improve patient comfort. Moreover, removal
of the patch backing may allow for verification that the intended
payload is delivered to the sample and/or subject by ensuring that
none of the therapeutic (or what amount of therapeutic) is present
on the breakable support after patch administration.
[0108] In a further aspect, the microneedles comprise breakable
supports. As used herein, "breakable" is capable of being
broken.
[0109] In various aspects, breakable is via a breakable support.
Thus, in various aspects a microneedle sidewall may comprise at
least one breakable support. For example, the support may resist
breaking under application of a normal force, but allow separation
through torsion, shearing, or other energy inputs. In a still
further aspect, the microneedles comprise a sidewall comprising a
breakable support.
[0110] In various aspects, breakable is via a perforation such as,
for example, a physical perforation or a chemical perforation.
[0111] In a further aspect, the microneedles comprise a perforated
sidewall. As used herein, a "perforation" refers to a specific
plane within the microneedle that is chemically or physically
distinct from the remainder of the array. In this way, one part of
the microneedle (e.g., the tip) may be separated from the rest of
the microneedle (e.g., the base). In various aspects, a perforation
includes a hole or slit. In various aspects a microneedle sidewall
may comprise at least one perforation.
[0112] In a further aspect, the sidewall is physically perforated.
For example, a sidewall may be physically perforated by computer
design.
[0113] In a further aspect, the sidewall is chemically perforated.
For example, a sidewall may be chemically perforated by a water
soluble material. Thus, in various aspects, chemically perforated
may be dissolvable. In a further aspect, chemically perforated may
be mechanically distinct.
[0114] Microneedles that can be mechanically or chemically
fragmented or removed from the backing are useful in that they
allow for rapid administration of therapeutics that have long term
drug release without the long term patch application. For example,
if a needle patch composition is designed to release drug over a
period of one week, then breakable microneedles could be applied to
the skin, fragmented, and the patch backing removed, with the
microneedle fragments embedded in the skin to release drug. This
could afford patients the benefit of long-term drug delivery
without the need to wear a patch for the entire duration of
therapy. Below are examples of breakable microneedle designs that
can be formed from additive manufacturing as taught herein.
[0115] a. Breakable Microneedles Having Physical Supports
[0116] In a further aspect, the microneedles comprise a sidewall
comprising a breakable support. The breakable support may be made
up of the same material as the rest of the microneedle.
Alternatively, the breakable support may be made up of a different
material than the rest of the microneedle.
[0117] Thus, in various aspects, microneedles are formed with
supports that resist breaking under application of a normal force
to the patch backing, but allow separation through torsion,
shearing, or other energy inputs (sound, heat, light, pressure)
(FIG. 1, panels A-I). This design would leave a portion(s) of the
needle embedded in the skin after patch removal depending on where
the breakable supports are positioned.
[0118] In a further aspect, of from about 0.1% to about 99% of the
sidewall comprises a breakable support. In a still further aspect,
of from about 0.1% to about 90% of the sidewall comprises a
breakable support. In yet a further aspect, of from about 0.1% to
about 75% of the sidewall comprises a breakable support. In an even
further aspect, of from about 0.1% to about 50% of the sidewall
comprises a breakable support. In a still further aspect, of from
about 0.1% to about 25% of the sidewall comprises a breakable
support. In yet a further aspect, of from about 0.1% to about 10%
of the sidewall comprises a breakable support. In an even further
aspect, of from about 0.1% to about 5% of the sidewall comprises a
breakable support. In a still further aspect, of from about 0.1% to
about 1% of the sidewall comprises a breakable support. In yet a
further aspect, of from about 1% to about 99% of the sidewall
comprises a breakable support. In an even further aspect, of from
about 5% to about 99% of the sidewall comprises a breakable
support. In a still further aspect, of from about 10% to about 99%
of the sidewall comprises a breakable support. In yet a further
aspect, of from about 25% to about 99% of the sidewall comprises a
breakable support. In an even further aspect, of from about 50% to
about 99% of the sidewall comprises a breakable support. In a still
further aspect, of from about 75% to about 99% of the sidewall
comprises a breakable support. In yet a further aspect, of from
about 90% to about 99% of the sidewall comprises a breakable
support.
[0119] In a further aspect, all of the microneedles on the array
comprise a sidewall comprising a breakable support. In a still
further aspect, at least about 75% of the microneedles on the array
comprise a sidewall comprising a breakable support. In yet a
further aspect, at least about 50% of the microneedles on the array
comprise a sidewall comprising a breakable support. In an even
further aspect, at least about 25% of the microneedles on the array
comprise a sidewall comprising a breakable support. In a still
further aspect, at least about 15% of the microneedles on the array
comprise a sidewall comprising a breakable support. In yet a
further aspect, at least about 10% of the microneedles on the array
comprise a sidewall comprising a breakable support. In an even
further aspect, at least about 5% of the microneedles on the array
comprise a sidewall comprising a breakable support. In a still
further aspect, a single microneedle on the array comprises a
sidewall comprising a breakable support.
[0120] b. Breakable Microneedles Having Physical Perforations
[0121] In a further aspect, the microneedles comprise a physically
perforated sidewall. Thus, in various aspects, microneedles are
formed with physical perforated sidewalls that resist breaking
under application of a Normal force to the patch backing, but allow
separation through torsion, shearing, or other energy inputs
(sound, heat, light, pressure) (FIG. 2, panels A-G). This design
differs from the support model in that tiny portions of the needle
would be omitted through computer design to make perforations in
the existing needle structure rather than adding material in the
form of supports. The effect would be similar in that portion(s) of
the needle would remain embedded in the skin after patch removal
depending on where the perforations were designed.
[0122] In a further aspect, of from about 0.1% to about 99% of the
sidewall is physically perforated. In a still further aspect, of
from about 0.1% to about 90% of the sidewall is physically
perforated. In yet a further aspect, of from about 0.1% to about
75% of the sidewall is physically perforated. In an even further
aspect, of from about 0.1% to about 50% of the sidewall is
physically perforated. In a still further aspect, of from about
0.1% to about 25% of the sidewall is physically perforated. In yet
a further aspect, of from about 0.1% to about 10% of the sidewall
is physically perforated. In an even further aspect, of from about
0.1% to about 5% of the sidewall is physically perforated. In a
still further aspect, of from about 0.1% to about 1% of the
sidewall is physically perforated. In yet a further aspect, of from
about 1% to about 99% of the sidewall is physically perforated. In
an even further aspect, of from about 5% to about 99% of the
sidewall is physically perforated. In a still further aspect, of
from about 10% to about 99% of the sidewall is physically
perforated. In yet a further aspect, of from about 25% to about 99%
of the sidewall is physically perforated. In an even further
aspect, of from about 50% to about 99% of the sidewall is
physically perforated. In a still further aspect, of from about 75%
to about 99% of the sidewall is physically perforated. In yet a
further aspect, of from about 90% to about 99% of the sidewall is
physically perforated.
[0123] In a further aspect, all of the microneedles on the array
comprise a physically perforated sidewall. In a still further
aspect, at least about 75% of the microneedles on the array
comprise a physically perforated sidewall. In yet a further aspect,
at least about 50% of the microneedles on the array comprise a
physically perforated sidewall. In an even further aspect, at least
about 25% of the microneedles on the array comprise a physically
perforated sidewall. In a still further aspect, at least about 15%
of the microneedles on the array comprise a physically perforated
sidewall. In yet a further aspect, at least about 10% of the
microneedles on the array comprise a physically perforated
sidewall. In an even further aspect, at least about 5% of the
microneedles on the array comprise physically perforated sidewall.
In a still further aspect, a single microneedle on the array
comprises a physically perforated sidewall.
C. BREAKABLE MICRONEEDLES HAVING CHEMICAL PERFORATIONS
[0124] In a further aspect, the microneedles comprise a chemically
perforated sidewall. Thus, in various aspects, microneedles are
formed with chemical perforations that can break down in the skin,
allowing for separation through physical (e.g., dissolving,
swelling, or cracking), chemical (e.g., pH or oxidation) or
enzymatic breakdown (biologically triggered) (FIG. 3, panels A-G).
This design would leave a portion(s) of the needle embedded in the
skin after patch removal depending on where the chemical
perforation is positioned.
[0125] In a further aspect, chemically perforated is via a
chain-transfer agent. For example, a chain-transfer agent may
reduce the size of the polymer chain. Without wishing to be bound
by theory, the chain-transfer agent may improve the solubility of
the polymer chain. In a still further aspect, the chain-transfer
agent is selected from N-acetyl cysteine, cysteine, dithiothreitol,
2-mercaptoethanol, isopropanol, and ethanol.
[0126] In a further aspect, of from about 0.1% to about 99% of the
sidewall is chemically perforated. In a still further aspect, of
from about 0.1% to about 90% of the sidewall is chemically
perforated. In yet a further aspect, of from about 0.1% to about
75% of the sidewall is chemically perforated. In an even further
aspect, of from about 0.1% to about 50% of the sidewall is
chemically perforated. In a still further aspect, of from about
0.1% to about 25% of the sidewall is chemically perforated. In yet
a further aspect, of from about 0.1% to about 10% of the sidewall
is chemically perforated. In an even further aspect, of from about
0.1% to about 5% of the sidewall is chemically perforated. In a
still further aspect, of from about 0.1% to about 1% of the
sidewall is chemically perforated. In yet a further aspect, of from
about 1% to about 99% of the sidewall is chemically perforated. In
an even further aspect, of from about 5% to about 99% of the
sidewall is chemically perforated. In a still further aspect, of
from about 10% to about 99% of the sidewall is chemically
perforated. In yet a further aspect, of from about 25% to about 99%
of the sidewall is chemically perforated. In an even further
aspect, of from about 50% to about 99% of the sidewall is
chemically perforated. In a still further aspect, of from about 75%
to about 99% of the sidewall is chemically perforated. In yet a
further aspect, of from about 90% to about 99% of the sidewall is
chemically perforated.
[0127] In a further aspect, all of the microneedles on the array
comprise a chemically perforated sidewall. In a still further
aspect, at least about 75% of the microneedles on the array
comprise a chemically perforated sidewall. In yet a further aspect,
at least about 50% of the microneedles on the array comprise a
chemically perforated sidewall. In an even further aspect, at least
about 25% of the microneedles on the array comprise a chemically
perforated sidewall. In a still further aspect, at least about 15%
of the microneedles on the array comprise a chemically perforated
sidewall. In yet a further aspect, at least about 10% of the
microneedles on the array comprise a chemically perforated
sidewall. In an even further aspect, at least about 5% of the
microneedles on the array comprise chemically perforated sidewall.
In a still further aspect, a single microneedle on the array
comprises a chemically perforated sidewall.
[0128] 2. Undercut Microneedle Structures
[0129] In a further aspect, the microneedles comprise an undercut
sidewall.
[0130] Microneedle insertion into the skin has been modeled in
three distinct phases: insertion, penetration and relaxation.
Briefly, the insertion phase spans the period of time from initial
contact with the skin until skin breach, penetration spans the time
from skin breach until the time at which the maximum penetration
depth is reached, and relaxation encompasses the period of time
from when the applied insertion force is removed until the needle
has reached steady state. In this relaxation phase, the skin's
natural elastic properties typically push needles back out of the
skin. While some studies have focused on decreasing forces of
insertion, few attempts have been made to prevent microneedles from
relaxing out of the skin. Utilizing additive manufacturing to make
arrowhead or other "undercut" microneedles provides the opportunity
to produce microneedles that remain deep within the skin at a
specified penetration depth.
[0131] Exemplary structural aspects may be found in, for example,
patents EP 1 465 535 B1, WO 2008053481 A1, US 2014/0005606 A1,
WO2012100002 A1 and the publication "Separable Arrowhead
Microneedles" (Chu et. al., Journal of Controlled Release,
149(3):242-249, 2011). O2012100002 A1, US20140170299, and EP 1 465
535 B1 contain undercut microneedles that are produced using
"in-plane" and "out-of-plane" techniques, wherein a two dimensional
sheet is cut and may then be folded to produce a semi-three
dimensional structure. However, each microneedle produced using
such techniques is a flat structure that exists within a single
spatial plane. A three-dimensional microneedle cannot be produced
using "in-plane" and "out-of-plane" approaches. Flat, semi-three
dimensional structures such as the folded arrowhead structures in
patent US20140170299 are likely to be weaker than a true
three-dimensional structure comprised of an equivalent material
because of these mechanical limitations. These techniques are
typically used to fabricate metal microneedles, which may pose
immunological hazards to patients. Further, the semi-three
dimensional structure produced using previous techniques limits
available cargo loading volume. Chu et. al. demonstrate the ability
to produce water soluble arrowhead microneedles using a two-step
process, wherein the microneedle tip is filled with a mixture of
PVP and PVA. An array of metal shafts are then manually inserted
into this mixture to create an arrowhead microneedle. This
technique is a multi-step manual batch process that utilizes
non-biocompatible materials. In US 2014/0005606 A1, a molding-based
method of producing purely biodegradable microneedles is proposed.
It is unclear, however, how such undercut structures are removed
from a mold. This molding-based process is also subject to the
lengthy fabrication times mentioned previously.
[0132] 3. Tiered Microneedles
[0133] In a further aspect, the microneedles are tiered. As used
herein, "tiered" refers to an array of microneedles containing
microneedles of multiple different heights. Thus, in various
aspects, microneedle patches are provided in which microneedles of
multiple different heights are included on a single array ("Tiered
Microneedles"). Without wishing to be bound by theory, these
microneedles may counteract the "bed-of-nails" effect, in which the
force of insertion on a microneedle array is distributed among the
individual needles in the array, thereby limiting the total amount
of force that can be applied to a single needle (FIG. 4A). Using
CLIP to produce an array in which multiple microneedle heights are
represented on a single patch (FIG. 4B) enables this force to be
concentrated on a smaller number of needles at a given time,
thereby reducing the total force required for insertion (Table 1).
Ideally, these microneedles would enable facile insertion into the
skin using small forces, such as the force of thumb. Without
wishing to be bound by theory, small insertion forces should allow
microneedles to be fabricated from a wider variety of materials and
to insert more deeply into the skin.
TABLE-US-00001 TABLE 1 Number of Layers Force Required for
Insertion* 1 F 3 F/3 4 F/4 n F/n *assumes equal number of needles
per tier
[0134] In various aspects, a tiered array may comprise microneedles
at two or more different heights. In a further aspect, a tiered
array may comprise microneedles at two or more different heights.
In a still further aspect, a tiered array may comprise microneedles
at three or more different heights. In yet a further aspect, a
tiered array may comprise microneedles at four or more different
heights. In an even further aspect, a tiered array may comprise
microneedles at five or more different heights. In a still further
aspect, a tiered array may comprise microneedles at six or more
different heights. In yet a further aspect, a tiered array may
comprise microneedles at seven or more different heights. In an
even further aspect, a tiered array may comprise microneedles at
eight or more different heights. In a still further aspect, a
tiered array may comprise microneedles at nine or more different
heights. In yet a further aspect, a tiered array may comprise
microneedles at ten or more different heights.
[0135] In a further aspect, the different heights may be arranged
randomly. In a still further aspect, the different heights may be
arranged from shortest to tallest. In yet a further aspect, the
different heights may be arranged via alternating rows. In an even
further aspect, the different heights may be arranged together such
as, for example, dividing the array in half (i.e., half one height
and half the other height).
[0136] In a further aspect, the microneedles may differ in height
by a ratio of at least about 1 to 100. In a still further aspect,
the microneedles may differ in height by a ratio of at least about
1 to 80. In yet a further aspect, the microneedles may differ in
height by a ratio of at least about 1 to 60. In an even further
aspect, the microneedles may differ in height by a ratio of at
least about 1 to 50. In a still further aspect, the microneedles
may differ in height by a ratio of at least about 1 to 40. In yet a
further aspect, the microneedles may differ in height by a ratio of
at least about 1 to 20. In an even further aspect, the microneedles
may differ in height by a ratio of at least about 1 to 10. In a
still further aspect, the microneedles may differ in height by a
ratio of at least about 1 to 5. In yet a further aspect, the
microneedles may differ in height by a ratio of at least about 1 to
2. In an even further aspect, the microneedles may differ in height
by a ratio of at least about 1 to 1.5. In a still further aspect,
the microneedles may differ in height by a ratio of at least about
1 to 1.05. In yet a further aspect, the microneedles may differ in
height by a ratio of at least about 1 to 1.005. In an even further
aspect, the microneedles may differ in height by a ratio of at
least about 1 to 1.0005.
[0137] 4. Microneedles with Curved or Discontinuous Sidewall
Profiles
[0138] In a further aspect, the microneedles comprise a curved or
discontinuous sidewall. In a still further aspect, the microneedles
comprise a curved sidewall. In yet a further aspect, the
microneedles comprise a discontinuous sidewall.
[0139] Some traditional microneedle fabrication techniques (such as
tilted UV photolithography) are limited to producing microneedles
with a straight sidewall profile. When square pyramidal
microneedles are used (shown in FIG. 5A) an inherent tradeoff
exists between reducing the force required to insert the needle and
improving needle strength. Due to these challenges, high aspect
ratio microneedles are subject to buckling.
[0140] Thus, in various aspects, microneedles are fabricated with
curved or discontinuous sidewall profiles (FIG. 5B-D). Without
wishing to be bound by the theory, the sharp, narrow tip may allow
for insertion into the skin with minimal force because the force
required for microneedle insertion decreases with tip sharpness.
Once the skin has been breached, the wider, more stable microneedle
could effectively insert into the skin without fracture.
[0141] 5. Microneedles with Complex X-Y Cross-Sections
[0142] In a further aspect, the microneedles comprise a
cross-section that is non-circular and non-polygonal. Thus, in
various aspects, microneedles are fabricated with complex X-Y
cross-sections. For example, microneedles may a cross-section that
is non-circular or non-polygonal (e.g., non-square, non-rectangle,
non-triangular, etc.) in shape in at least a part of the length
thereof. Exemplary non-circular and non-polygonal shapes include,
but are not limited to, a star, a cross, a heart, a cone, and a
partial sphere (e.g., moon).
[0143] Some common mechanical models correlate the amount of energy
required to insert a microneedle into the skin with the volume of
tissue that must be deformed during its insertion. Because it may
be desirable to insert microneedles into the skin at a specific
penetration depth (for targeting certain cell types, accessing the
bloodstream, etc.), it may be desirable to produce microneedles
that have limited volume for a given height, enabling the amount of
force required to insert the needle to a given depth to be reduced.
An example of such a design is shown in FIG. 6A. In this specific
example, a microneedle with "fins" is designed to maintain the
mechanical strength of a tall microneedle. The cutouts between fins
reduce the amount of tissue that needs to be deformed to
effectively insert this needle into the skin at a given depth. A
number of other similar microneedles may be fabricated. Exemplary
potential non-circular and non-polygonal cross-sections may
include, but are not limited to, those shown in FIG. 6B.
[0144] C. Methods of Making Microneedles with Additive
Manufacturing
[0145] In one aspect, disclosed are methods of making a disclosed
microneedle device.
[0146] In one aspect, disclosed are methods of making a microneedle
device, the method comprising the steps of: (a) providing a build
elevator and an optically transparent build surface, wherein the
build elevator and the build surface together define a build region
there between, wherein the build surface is permeable to a
polymerization inhibitor, and wherein the build surface is in fluid
communication with a source of the polymerization inhibitor; (b)
filling the build region with a polymerizable liquid; (c)
irradiating the build region through the build surface to produce a
solid polymerized region in the build region; (d) forming or
maintaining a liquid film release layer between the solid
polymerized region and the build surface, wherein the liquid film
release layer comprises the polymerizable liquid, and wherein the
polymerization of the liquid is inhibited by the polymerization
inhibitor; and (e) advancing the build elevator away from the build
surface to create a subsequent build region between the solid
polymerized region and the build surface while concurrently filling
the subsequent build region with the polymerizable liquid, wherein
the device comprises: (f) a backing; and (g) a plurality of
biocompatible microneedles projecting from the backing, wherein the
microneedles comprise one or more of: (i) a curved, discontinuous,
undercut, or perforated sidewall; (ii) a sidewall comprising a
breakable support; and (iii) a cross-section that is non-circular
and non-polygonal, and/or wherein the microneedles are tiered,
thereby making the microneedle device.
[0147] In one aspect, disclosed are methods of making a disclosed
microneedle device, comprising the steps of: (a) providing a build
elevator and an optically transparent build surface defining a
build region there between, said build surface being permeable to a
polymerization inhibitor, and with said build surface in fluid
communication with a source of the polymerization inhibitor; (b)
filling said build region with a polymerizable liquid, said
polymerizable liquid contacting said build surface; (c) irradiating
said build region through said build surface to produce a solid
polymerized region in said build region, while forming or
maintaining a liquid film release layer comprised of said
polymerizable liquid formed between said solid polymerized region
and said build surface, wherein the polymerization of which liquid
film is inhibited by said polymerization inhibitor; and (d)
advancing said build elevator with said polymerized region adhered
thereto away from said build surface to create a subsequent build
region between said polymerized region and said build surface while
concurrently filling said subsequent build region with
polymerizable liquid as in step (b), to thereby form said
microneedle device. In a further aspect, the method further
comprises (e) continuing and/or repeating steps (c) and (d) to
produce a subsequent polymerized region adhered to a previous
polymerized region until the continued or repeated deposition of
polymerized regions adhered to one another forms said microneedle
device. In a still further aspect, steps (c) and (d) are carried
out concurrently.
[0148] Aspects of the polymeric microneedles as taught herein may
be fabricated with a continuous liquid interface printing (CLIP)
apparatus, including, but not limited, to those described in PCT
application publication WO 2014/126837 to DeSimone et al., the
contents of which are incorporated by reference herein in its
entirety. A representative diagram of the CLIP process is shown in
FIG. 7A. A photopolymerizable liquid is illuminated with radiation
in a shape defined by a computational file (.svg, .ctl, bitmap,
etc.) of a microneedle patch. As the photopolymerizable liquid is
exposed to radiation, the microneedle patch attaches to build
elevator 4, which pulls the part through the liquid resin with a
substantially continuous upward movement. Unlike other additive
manufacturing approaches, where separation, recoating, and
repositioning steps are required between each sequential layer,
CLIP enables continuous (i.e., not layer-by-layer) generation of
the part through the generation of a liquid "dead zone" at the
interface between the permeable build surface and the building
microneedle patch. Without wishing to be bound by theory, the dead
zone may be created when oxygen, which acts as a polymerization
inhibitor, passes through the oxygen-permeable build surface.
Because photopolymerization cannot occur in the oxygen containing
region ("dead zone"), this region remains fluid, and the building
part does not physically attach to the build surface. Irradiation
may be generated using, for example, a UV LED light source
reflecting off of a digital micromirror device.
[0149] Two examples of CLIP printers and their respective
dimensions are shown in FIG. 7B and FIG. 7C and Table 1. These two
instruments serve as examples of CLIP printers, but a number of
different alternative systems could be utilized. For example, while
air is utilized as a photopolymerization inhibitor in these
systems, an alternative inhibitor such as pure oxygen or ammonia
could also be used. Furthermore, while these systems use
ultraviolet light, a number of alternative light sources with
irradiation at any point on the electromagnetic spectrum (visible,
nearIR, gamma radiation, etc.) could be utilized.
[0150] Selection of microneedle compositions may be based on a
number of different factors including, but not limited to,
solubility, biocompatibility, swelling or degradation kinetics, or
mechanical properties, among many others. Exemplary microneedles
have been fabricated from a number of compositions such as
polyethylene glycol, polyacrylic acid, and polycaprolactone, as
shown in FIG. 8A-C.
[0151] A number of other materials could also be utilized for the
formation of microneedles as taught herein. Some examples include,
but are not limited to, polyesters (polycaprolactone, polyglycolic
acid, polylactic acid, polylactic-co-glycolic acid), polyethers
(polyethylene glycol), thiol-enes, anhydrides, acrylate polymers
(polyacrylic acid, poly methylmethacrylate), vinyl polymers
(polyvinyl alcohol, polyvinylpyrrolidone, vinyl carbonates, vinyl
esters), acrylamides, natural polymers (hyaluronic acid, chitosan,
collagen, gelatin, carboxymethylcellulose), etc. Blends or
copolymers of aforementioned materials may be generated to tune
material properties. Any of these materials may or may not be
functionalized with photoreactive groups. A blend of photoreactive
monomer with a non-photoreactive monomer may be utilized, for
example, to generate porous needles.
[0152] In various aspects, the disclosed microneedles may contain
two or more different sections of spatially distinct compositions
(as opposed to a copolymer blend). Differences in composition may
include, for example, a difference in the type of monomer or
polymer utilized in the formulation, a difference in loading of the
therapeutic, a difference in crosslinking density of the final
needle, a difference in coating of the final microneedle, or any
other chemical differences in the liquid resin.
[0153] For example, microneedles with different layers may be
fabricated. See, e.g., U.S. Pat. No. 8,734,697, US 2011/0028905 A1,
US 2011/0152792, U.S. Pat. No. 8,491,534 and US 2008/0269685. All
of these patents utilize a mold-filling method to generate
microneedles with multiple layers. Briefly, a microneedle master
(typically made of metal or silicon) is used to generate a
polymeric mold, often cast in PDMS. This mold is then partially
filled with a specified quantity of the first material with a
series of time consuming vacuum and centrifugation steps. After
drying, the remainder of the mold is filled with a second material
of interest.
[0154] However, the disclosed microneedles may be accomplished with
the methods of additive manufacturing. Utilizing an additive
manufacturing process for the production of microneedles enables
microneedles to be produced more quickly than mold-based
techniques. Microneedles may also be produced in shapes that cannot
be molded, such as, for example, undercut microneedles. Additive
manufacturing also has the opportunity to increase consistency in
microneedle composition (microneedle dimensions, drug-loading, tip
sharpness, etc.) across an array by automating the production
process.
[0155] FIG. 9 shows a representative CLIP process that could be
utilized to produce multi-component microneedles wherein components
are spatially segmented along the z axis of the needle. Briefly,
the CLIP method shown in FIG. 7A can be utilized to fabricate a
portion of the microneedle patch from a resin of resin composition
A 10. Build elevator 4 is then lifted above the build platform in
step 2 and the liquid resin pool is removed and replaced with a new
resin pool of resin composition B 11. The remainder of the
microneedle is then fabricated with resin composition B.
[0156] The technique demonstrated in FIG. 7A could be utilized to
produce multi-component microneedle patches of a number of
different aspects, depicted in FIG. 10A-D. These aspects can be
utilized for a variety of different purposes. For example, the
aspect shown in FIG. 10A is a microneedle in which the needle tip
is a different composition than the remainder of the needle. In
some cases, the therapeutic would be loaded into the microneedle
tip, but not be loaded into the base. Without wishing to be bound
by theory, this may be useful for maximizing the amount of a
therapeutic that is delivered to the skin if the needle does not
completely insert into the skin. This high delivery efficiency is
particularly useful for therapeutics that are expensive or have
limited availability, where a dose-sparing effect is desirable.
Localizing the therapeutic at the tip may also enable more
consistent delivery in cases where different clinicians have
different insertion techniques, which result in differences in
penetration depth of the needle between applications.
[0157] Furthermore, this technique enables therapeutic to be
delivered to a specific layer within the skin (e.g., stratum
cornerum, epidermis, dermis). Without wishing to be bound by
theory, layer-specific delivery may enable localization of the
therapeutic to a specific cell type or allow for more control over
the rate of diffusion of this therapeutic from the site of
insertion into the skin to the bloodstream.
[0158] In other aspects, both the base and tip may be loaded with
therapeutic. Compositions with different release/degradation
kinetics may enable further control over pharmacokinetic profiles.
For example, one composition could enable rapid burst release of
the therapeutic, while the other composition could provide
sustained release of the therapeutic out of the needle.
[0159] In other aspects, the tip and base compositions could be
chosen for differing mechanical properties, wherein a strong
material could be used for the tip and a weaker material (which is
desirable for another property, such as its drug solubility or
release characteristics) could be utilized for the base. In this
way, the desired chemical and mechanical properties of the needle
could be obtained by combining the properties of each individual
matrix.
[0160] Other aspects include, but are not limited to, patches in
which the patch backing is a different material than the needles
(FIG. 10B), tip-loaded patches that contain a backing of a distinct
composition (FIG. 10C) and needles in which multiple layers are
present (FIG. 10D). These aspects may be utilized for any of the
purposes listed above. An additional use of patches which contain a
distinct backing composition is the ability to dissolve away a
water soluble backing layer to deposit needles within the skin.
[0161] In a further aspect, the method further comprises continuing
and/or repeating steps (c)-(e) to create a subsequent polymerized
region adhered to a previous polymerized region. This may be
continued, for example, until the continued or repeated deposition
of polymerized regions adhered to one another forms said
microneedle device.
[0162] In a further aspect, steps (c)-(e) are performed
simultaneously. In a still further aspect, steps (c)-(e) are
performed sequentially.
[0163] In a further aspect, the polymerizable liquid comprises a
free radical polymerizable liquid and the polymerization inhibitor
comprises oxygen. In a still further aspect, the polymerizable
liquid comprises an acid-catalyzed or cationically polymerizable
liquid and the polymerization inhibitor comprises a base.
[0164] In a further aspect, irradiating is via actinic
radiation.
[0165] In a further aspect, advancing is carried out at a
cumulative rate of at least 0.1, 1, 10, 100, or 1000 microns per
second. In a still further aspect, advancing is carried out at a
cumulative rate of at least 0.1, 1, 10, or 100 microns per second.
In yet a further aspect, advancing is carried out at a cumulative
rate of at least 0.1, 1, or 10 microns per second. In an even
further aspect, advancing is carried out at a cumulative rate of at
least 0.1 or 1 microns per second. In a still further aspect,
advancing is carried out at a cumulative rate of at least 1, 10,
100, or 1000 microns per second. In yet a further aspect, advancing
is carried out at a cumulative rate of at least 10, 100, or 1000
microns per second. In an even further aspect, advancing is carried
out at a cumulative rate of at least 100 or 1000 microns per
second.
[0166] In a further aspect, advancing comprises moving the build
elevator vertically from the build surface.
[0167] In a further aspect, the microneedle device is formed in
less than 30 minutes, less than 20 minutes, or less than 10
minutes. In a still further aspect, the microneedle device is
formed in less than 30 minutes. In yet a further aspect, the
microneedle device is formed in less than 25 minutes. In an even
further aspect, the microneedle device is formed in less than 20
minutes. In a still further aspect, the microneedle device is
formed in less than 15 minutes. In yet a further aspect, the
microneedle device is formed in less than 10 minutes. In an even
further aspect, the microneedle device is formed in less than 5
minutes.
D. METHODS OF DELIVERING THERAPEUTIC AGENTS
[0168] In one aspect, disclosed are methods of delivering a
therapeutic agent to a subject, the method comprising administering
to the subject a microneedle device comprising: (a) a backing; and
(b) a plurality of biocompatible microneedles projecting from the
backing, wherein the microneedles comprise a therapeutic agent and
one or more of: (i) a curved, discontinuous, undercut, or
perforated sidewall; (ii) a sidewall comprising a breakable
support; and (iii) a cross-section that is non-circular and
non-polygonal, and/or wherein the microneedles are tiered, thereby
delivering the therapeutic agent. Thus, in various aspects, the
disclosed microneedle devices could be utilized for delivery of
therapeutic agents useful for a number of different therapeutic
indications. Cargos may include, for example, nano- or
micro-particles, proteins, small molecules, enzymes, sugars, and
nucleic acids, etc. See also Kim et al., "Microneedles for drug and
vaccine delivery," Adv. Drug Deliv. Rev. 64(14):1547-1568, 2012;
Indermun et al., "Current advances in the fabrication of
microneedles for transdermal delivery," J Controlled Release
185:130-138, 2014.
[0169] Exemplary agents and indications include, but are not
limited to, delivery of insulin for diabetes, delivery of
chemotherapeutics or vaccines for cancer, e.g., skin cancer
(melanoma, basal cell carcinoma, inflammatory breast cancer, etc.),
delivery of anesthetics, delivery of cosmeceutical agents such as
botox, delivery of anticoagulants such as heparin, delivery of
various enzymes (such as butyrylcholinesterase) and growth
hormones, delivery of immunomodulatory agents for treating
autoimmune diseases such as psoriasis, bullous pemphigoid,
epidermolysis bullosa, dermatomyositis, scleroderma, eczema,
rheumatoid arthritis, and multiple sclerosis, etc. Microneedles
also may be used for vaccines (e.g., particulate, protein subunit,
nucleic acid, or whole pathogen) for a number of different
infectious or non-infectious diseases. Microneedles also may be
utilized to diagnose a variety of conditions such as, for example,
diabetes, heart attacks, infectious diseases, and bacterial
infections, or to perform a standard blood test.
[0170] In a further aspect, the microneedle devices described
herein can be used for cutaneous immunization.
[0171] In a further aspect, the microneedle devices can be used for
chemotherapy and immunochemotherapy applications, for example, as
an alternative to or in addition to traditional topical
chemotherapy approaches. In the case of cutaneous tumors, including
skin derived tumors (e.g., basal cell, squamous cell, Merkel cell,
and melanomas) and tumors metastatic to skin (e.g., breast cancer
and melanoma), topical delivery may be desired. Current methods of
topical delivery generally require the application of creams or
repeated local injections. The effectiveness of these approaches is
currently limited by limited penetration of active agents into the
skin, non-specificity, and unwanted side effects.
[0172] Further, multiple bioactive agents can be delivered in a
single microneedle array (e.g., a patch). This enables an
immunochemotherapeutic approach based on the co-delivery of a
cytotoxic agent with and immune stimulant (adjuvants). In an
immunogenic environment created by the adjuvant, tumor antigens
releases from dying tumor cells will be presented to the immune
system, inducing a local and systemic anti-tumor immune response
capable of rejecting tumor cells at the site of the treatment and
throughout the body. See also U.S. Pat. No. 8,834,423 to Falo et
al.
[0173] In a further aspect, the therapeutic agent comprises a
protein therapeutic, a small molecule therapeutic, a vaccine
antigen, or an antigenic fragment thereof.
E. METHODS OF TREATING A DISEASE IN A SUBJECT
[0174] In one aspect, disclosed are methods of treating a disease
in a subject, the method comprising administering to the subject a
microneedle device comprising: (a) a backing; and (b) a plurality
of biocompatible microneedles projecting from the backing, wherein
the microneedles comprise a therapeutic agent and one or more of:
(i) a curved, discontinuous, undercut, or perforated sidewall; (ii)
a sidewall comprising a breakable support; and (iii) a
cross-section that is non-circular and non-polygonal, and/or
wherein the microneedles are tiered, thereby treating the
disease.
[0175] In one aspect, disclosed are methods of treating a disease
or condition in a subject in need thereof, comprising administering
to the subject a disclosed microneedle device.
[0176] Thus, in various aspects, the disclosed microneedle devices
may be useful for the delivery of therapeutic agents useful for
treatment a number of different therapeutic indications.
[0177] "Treat" or "treatment" as used herein refers to any type of
treatment that imparts a benefit to a subject afflicted with a
disease or disorder that may benefit from microneedle delivery of a
therapeutic agent, including improvement in the condition of the
patient (e.g., in one or more symptoms), delay in the progression
of the disease or disorder, delay in onset or recurrence of the
disease or disorder, etc.
[0178] For example, microneedle devices as taught herein may be
used to deliver insulin in the treatment of diabetes. See Fukushima
et al., "Pharmacokinetic and Pharmacodynamic Evaluation of Insulin
Dissolving Microneedles in Dogs," Diabetes Technology &
Therapeutics 12(6):465-474, 2010; Ito et al., "Transdermal Insulin
Application System with Dissolving Microneedles," Diabetes
Technology & Therapeutics 14(10):891-899, 2012; Ito et al.,
"Two-layered dissolving microneedles formulated with
intermediate-acting insulin," International J Pharmaceutics
436:387-393, 2012; Ling et al., "Dissolving polymer microneedle
patches for rapid and efficient transdermal delivery of insulin to
diabetic rats," Acta Biomaterialia 9:8952-8961, 2013.
[0179] The present invention is primarily concerned with the
treatment of human subjects, but the invention may also be carried
out on animal subjects, particularly mammalian subjects such as
mice, rats, dogs, cats, livestock and horses for veterinary
purposes, and for drug screening and drug development purposes.
Subjects may be of any age, including infant, juvenile, adolescent,
adult, and geriatric subjects.
[0180] The ability to easily modulate patch size and shape can be
used to make personalized microneedle patches. For example, a
microneedle patch could be made to specifically cover the area of
an individual's wound or to alter the size of a microneedle patch
to match the dosage of a medication to a patient's body weight,
etc. Additive manufacturing methods also provide the opportunity to
produce microneedle patches at the point of care.
[0181] In a further aspect, the therapeutic agent comprises a
protein therapeutic or a small molecule therapeutic.
[0182] In a further aspect, the disease is diabetes and the
therapeutic agent is insulin. In a still further aspect, the
disease is a bacterial infection and the therapeutic agent is an
antibiotic. In yet a further aspect, the disease is cancer and the
therapeutic agent is a chemotherapeutic agent.
F. METHODS OF DETECTING A BIOMARKER IN A SAMPLE
[0183] In one aspect, disclosed are methods of detecting a
biomarker in a sample, the method comprising: (a) providing a
microneedle device comprising: (i) a backing; and (ii) a plurality
of biocompatible microneedles projecting from the backing, wherein
the microneedles comprise a probe for the biomarker and one or more
of: (1) a curved, discontinuous, undercut, and/or perforated
sidewall; (2) a sidewall comprising a breakable support; and (3) a
cross-section that is non-circular and non-polygonal, and/or
wherein the microneedles are tiered; (b) contacting the device with
the sample; and (c) identifying the biomarker, thereby detecting
the biomarker in the sample.
[0184] In a further aspect, contacting is during an intraoperative
procedure. In a still further aspect, contacting is after the
sample has been removed from the subject.
[0185] In a further aspect, the sample is a tissue or biological
sample. In a still further aspect, the sample is a tissue sample.
In yet a further aspect, the sample is a biological sample. The
tissue or biological sample can be from an organ such as, for
example, a brain, a heart, a breast, a liver, a pancreas, a spleen,
a bladder, a stomach, a lung, a uterus, a cervix, a prostate, a
kidney, an intestine, an appendix, and a colon.
[0186] In a further aspect, the sample is in a mammal. In a still
further aspect, the mammal is human.
[0187] The probe may be covalently attached to a microneedle.
Alternatively, the probe may be non-covalently attached to a
microneedle. Examples of probes include, but are not limited to,
polynucleotides, polypeptides, proteins, antibodies, small
molecules, and biological receptors.
[0188] The present invention is explained in greater detail in the
following non-limiting examples.
G. EXAMPLES
[0189] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how the devices and/or methods claimed herein are
made and evaluated, and are intended to be purely exemplary of the
invention and are not intended to limit the scope of what the
inventors regard as their invention. Efforts have been made to
ensure accuracy with respect to numbers (e.g., amounts,
temperature, etc.), but some errors and deviations should be
accounted for. Unless indicated otherwise, parts are parts by
weight, temperature is in .degree. C. or is at ambient temperature,
and pressure is at or near atmospheric.
[0190] 1. Materials
[0191] The ramp test patterns in FIG. 11C were printed with
trimethylolpropane triacrylate (TMPTA) using the photoinitiator,
diphenyl (2,4,6-trimethyl-benzoyl)phosphine oxide. Other objects
were printed with a combination of monomers from Sartomer (CN2920
& CN981), TMPTA, and reactive diluents such as
n-vinylpyrrolidone, isobornyl acrylate, and cyclohexane dimethanol
di-vinyl ether. Photoinitiators,
phenylbis(2,4,6-trimethyl-benzoyl)phosphine oxide,
1-hydroxycyclohexyl phenyl ketone, and
2-benzyl-2-(dimethylamino)-4'-morpholinobutyrophenone, and an
assortment of dyes from Wikoff and Mayzo were also utilized.
[0192] 2. Continuous Liquid Interface Printing
[0193] FIG. 11A illustrates the simple architecture and operation
of a 3D printer that takes advantage of an oxygen inhibited dead
zone. As shown in FIG. 11A, the part (gyroid) is printed
continuously by simultaneously elevating the build support plate
while changing the 2-D cross-sectional UV images from the imaging
unit. The oxygen permeable window creates a dead zone (persistent
liquid interface) between the elevating part and window.
[0194] CLIP proceeds via projecting a continuous sequence of UV
images (generated by a digital light processing imaging unit)
through an oxygen-permeable, UV-transparent window below a liquid
resin bath. The dead zone created above the window maintains a
liquid interface below the advancing part. Above the dead zone, the
curing part is continuously drawn out of the resin bath creating
suction forces that continually renew reactive liquid resin. This
continual process is fundamentally different from traditional
bottom-up stereolithography printers where UV exposure, resin
renewal, and part movement must be conducted in separate and
discrete steps (FIG. 12). Even for inverted top-down approaches
where photo-polymerization occurs at an air-resin interface (the
part is successively lowered into a resin bath during printing
(Gibson et al., Additive Manufacturing Technologies: Rapid
Prototyping to Direct Digital Manufacturing. (Springer, N.Y.,
2010); Jacobs, P. F., Rapid Prototyping & Manufacturing:
Fundamentals of StereoLithography. Society of Manufacturing
Engineers, Dearborn, MII, 1992)), these steps must be conducted
sequentially for the formation of each layer. Since each step takes
several seconds to implement for each layer, and since each layer
of a part has a typical thickness of 50-100 p.m, vertical print
speeds are restricted to a few mm/hr (Gibson et al., Additive
Manufacturing Technologies: Rapid Prototyping to Direct Digital
Manufacturing. (Springer, N.Y., 2010)). By contrast, the print
speed for CLIP is limited by resin cure rates and viscosity not by
step-wise layer formation. For example, the 5 cm tall gyroid and
argyle structures shown in FIG. 11B were printed at vertical draw
speeds 500 mm/hr, i.e., in less than 10 minutes. An additional
benefit of continuous printing is that choice of slicing thickness,
which affects part resolution, does not influence print speed as
shown in the ramp test patterns in FIG. 11C. Specifically, FIG. 11C
shows representative RAMP test patterns printed at the same print
speed regardless of slicing thickness (100 .mu.M, 25 .mu.M, and 1
.mu.M). Since CLIP is continuous, the refresh rate of projected
images may be increased without altering print speed, ultimately
allowing for parts to approach layerless 3D objects.
[0195] Referring to FIG. 12, traditional stereolithography first
exposes resin to UV light, which causes cured adhesion to a build
window such as glass. Next, the part must be mechanically
separated, followed by resin re-coating and part re-positioning,
before the next layer can be exposed. CLIP, with a permanent liquid
interface at the window, allows the part to be continuously exposed
while elevating, thereby eliminating three steps in the
process.
[0196] 3. Fabrication of Microneedles
[0197] To demonstrate the utility of CLIP in addressing challenges
associated with microneedle fabrication times, microneedles were
fabricated using trimethylolpropane triacrylate (TMPTA) plus 2.5 wt
% DPO. Briefly, a computer aided design (CAD) file was generated
and sliced using the open source software Slic3r in 1 .mu.m slices.
This file was printed using the CLIP7 (Table 2) with 5.4
mW/cm.sup.2 of light at 100 mm/hr. Environmental Scanning Electron
Micrographs of microneedles of three different heights along with
their dimensions and print times are illustrated in FIG. 19A-D and
Table 3.
TABLE-US-00002 TABLE 2 CLIP7 CLIP Mini Light Source (nM LED) 370
365 Max. Light Intensity (W/cm.sup.2) 77 30 Theoretical Resolution
(.mu.M) 10 20 Build Area (mm) ~7.7 .times. 15 .times. 24 .times.
100 10.3 .times. 100 Unit Size (inches) ~36 H .times. 181/4 H
.times. 9 15/16 12 W .times. 24 D W .times. 121/2 D
TABLE-US-00003 TABLE 3 Nominal Actual Nominal Actual Print Tip
Height Height Width Width Time Radius (.mu.M) (.mu.M) (.mu.M)
(.mu.M) (s) (.mu.M) 1000 1023.5 .+-. 52.8 333.3 321.4 .+-. 20.0
84.6 2.3 .+-. 0.5 700 712.6 .+-. 13.7 233.3 236.3 .+-. 8.7 79.5 3.1
.+-. 0.4 400 383.7 .+-. 8.6 133.3 135.4 .+-. 8.3 80.6
[0198] The example microneedles fabricated with CLIP are
particularly sharp based on measurement of tip radius. Because
insertion force is directly related to force of insertion, it is
hypothesized that less force would be required to insert these
needles into the skin than other polymeric microneedles.
[0199] 4. Microneedle Composition and Testing
[0200] Microneedles have been fabricated with CLIP from a number of
compositions, such as polyethylene glycol, polyacrylic acid, and
polycaprolactone, as shown in FIG. 8A-C. The resin compositions for
these needles are acrylic acid (Acros Organics, 99.5% purity), a
polycaprolactone trimethacrylate (Mn=900, synthesized by known
methods) and a poly (ethylene glycol) dimethacrylate (Mn=550,
Sigma), all mixed with 2.5 wt %
Diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide as a
photoinitiator. Microneedles were fabricated on the CLIP7 (see
Table 1) at 25 mm/hr with 8.3, 1.5, and 1.2 mW/cm.sup.2 of light
(.lamda.=370), for acrylic acid, polyethylene glycol, and
polycaprolactone, respectively. The fabrication of each patch took
approximately 5 minutes.
[0201] Monomers selected for CLIP microneedle fabrication were
chosen for the differing solubility and release characteristics of
their respective polymers. Poly(ethylene glycol) is a
water-miscible polymer previously used for microneedle fabrication
and encapsulation of hydrophilic cargos. Drug release from the PEG
hydrogel is likely to occur via diffusion out of the swellable
matrix, followed by slow degradation of the residual needle.
Poly(caprolactone) is a lipophilic material useful for
incorporating hydrophobic molecules (such as chemotherapeutics),
which typically exhibit poor oral bioavailability. It is
susceptible to hydrolytic degradation of ester linkages, enabling
sustained release of lipophilic cargo. Acrylic acid, which
undergoes precipitation polymerization to form linear polyacrylic
acid, was selected as a rapidly degrading hydrophilic matrix which
is expected to dissolve upon insertion into the skin.
[0202] Microneedles of three different compositions were applied to
ex vivo murine skin by pressing firmly on the back of the patch
with the thumb for 30 seconds. The ability of these microneedles to
insert into the skin was assessed using a green tissue marking dye,
which only stains live cells. The skin samples shown in FIG. 20A-D
show that TMPTA microneedles, polyacrylic acid microneedles, and
poly(ethylene glycol) microneedles effectively insert into the
skin.
[0203] Calculations presented below demonstrate that microneedles
are a feasible method for delivery of therapeutic proteins such as
insulin. Specifically, for a 4U dose of insulin, with an arrowhead
shape of the given dimensions (FIG. 21A and FIG. 21B), it is
estimated that a patch of 4.25 cm.sup.2-6.25 cm.sup.2 would be
required, depending on spacing between needles. A square patch
measuring 2.1-2.5 cm on each side would provide sufficient volume.
The calculations of the patch area are illustrated below:
V pyramid = 0.2 mm .times. 0.2 mm .times. 0.4 mm 3 = 0.0053 mm 3 V
rectangle = 0.2 mm .times. 0.2 mm .times. 0.4 mm = 0.016 mm 3 }
0.0213 mm 3 = 0.0213 ul volume / Needle Tip ##EQU00001##
[0204] 4 (IU) Insulin=145 .mu.g insulin=14.5 .mu.L insulin [10
.mu.g/.mu.L max solubility]
14.5 .mu.L volume/0.0213 .mu.L per needle=680 needles
Wide Needle Spacing (0.1 mm Between Needles)
Area/needle=0.09 mm.sup.2.times.680 needles=61.2 mm.sup.2=6.12
cm.sup.2
Narrow Needle Spacing (0.05 mm Between Needles)
Area/needle=0.0625 mm.sup.2.times.680 needles=42.5 mm.sup.2=4.25
cm.sup.2
[0205] Of course, alternative sizes, shapes and dimensions of the
microneedles and patch backing could also be used.
[0206] 5. Incorporation and Release of Biological Payloads from
Clip Fabricated Biocompatible Material
[0207] A. Incorporation of Vaccine Antigen into Clip Fabricated
Poly(Acrylic Acid) (PAA) Materials
[0208] Fluorescently labeled (Alexa Fluor.RTM. 647) model vaccine
antigen ovalbumin (referred to as OVA-647) was incorporated into a
PAA matrix using CLIP and a resin containing 97.5 wt % acrylic acid
monomer (Sigma), 2.3 wt % DPO photo initiator (Sigma), 8.97 wt %
water, 0.01 wt % OVA-647 (Invitrogen). Resin of an identical
composition, excluding the 0.01 wt % OVA-647, was used as a
negative control for OVA-647 incorporation. The shape of the CLIP
fabricated test parts was cylindrical with 3 mm diameter.times.3 mm
height, containing a hollowed out center 1.5 mm diameter (FIG. 22A
and FIG. 22B). Parts were fabricated using continuous printing on
the CLIP7 apparatus (Carbon 3D) with a drawspeed of 100 mm/hr and
8.4 mW/cm2 light intensity. OVA-647 incorporation into CLIP
fabricated PAA parts was confirmed using microscopy (FIG. 22C). The
OVA-647 and vehicle control PAA cylinders were dissolved in 2 mL of
water and the solution was then centrifuged at 20,000.times.g for
10 minutes to remove any insoluble material and OVA-647 release was
measured using a fluorescence plate reader. OVA-647 was readily
detected above background florescence levels from vehicle control
cylinders (FIG. 23), indicating that OVA-647 is released form PAA
cylinders dissolved in water. Without wishing to be bound by
theory, these results indicate that vaccine antigens can be
incorporated and released form CLIP fabricated biomaterials.
[0209] Referring to FIG. 23, vehicle PAA cylinders (containing 8.97
wt % water) or OVA-647 resin (0.09 wt % OVA-647 in water) were
tested. Each bar represents an independent cylinder dissolved in 2
mL of water and the supernatant was measured for OVA-647
fluorescence. Values were normalized to a standard curve of soluble
OVA-647. All measurements were performed in duplicate.
[0210] b. Incorporation of Active Vaccine Adjuvant into Clip
Fabricated Poly(Acrylic Acid) (PAA) Material
[0211] Resiquimod (R-848, Chemdea) was incorporated into test parts
with identical dimensions described in FIG. 22A using CLIP and a
resin containing 95.42 wt % acrylic acid monomer, 2.44 wt % DPO
photo initiator, 2.12 wt % DMSO, 0.02 wt % R-848. Resin of an
identical composition, excluding the 0.02 wt % R-848, was used as a
negative control for R-848 incorporation and release.
[0212] Parts were fabricated using continuous printing on the CLIP7
apparatus (Carbon 3D) with a drawspeed of 100 mm/hr and 8.4 mW/cm2
light intensity. CLIP fabricated PAA parts were washed for 16 hours
in acetone to remove residual resin and dried by desiccation. PAA
parts were then dissolved in 3 mL of water and the solution was
then centrifuged at 20,000.times.g for 10 minutes to remove any
insoluble material and R-848 bioactivity was measured using a
cellular based bioassay. Primary murine macrophages were stimulated
with supernatant from R-848 containing cylinders or vehicle control
cylinders dissolved in phosphate buffered saline (PBS, Sigma) water
for 8 hours and an Enzyme Linked Immunosorbant Assay (ELISA) method
was used to measure inflammatory cytokine IL-6 (ELISA kit from BD
Biosciences) release by macrophages, a well-established assay for
measuring innate immune responses to adjuvants. R-848 bio-activity
was readily detectable in the supernatant fraction of dissolved
CLIP fabricated PAA cylinders made with R-848 containing resin
(FIG. 24), whereas no activity is measured with the vehicle control
cylinders, suggesting that the macrophages were responding
specifically to the R-848 and not to the CLIP fabricated PAA
polymer itself. Without wishing to be bound by theory, these
results indicate that small molecule drugs have the capacity to
remain bioactive after incorporation and release from CLIP
fabricated biomaterials.
[0213] Referring to FIG. 24, cylinders were made with resin
containing 2.12 wt % DMSO (Vehicle Control) or 0.02 wt % R848 in
DMSO (R-848). Cylinders were washed in acetone, desiccated, and
dissolved in 3 mL of water and the supernatant was used to
stimulate bone marrow macrophages for 8 hours. Each bar represents
an independent cylinder. IL-6 secretion was measured in duplicate
by ELISA as a readout for R848 activity. Pos. Cntrl, soluble R848
(100 ng/mL).
[0214] C. Incorporation of Human Insulin (Insulin) into Clip
Fabricated Poly(Acrylic Acid) (PAA) Material
[0215] Insulin (Sigma) was incorporated into test parts with
identical dimensions described in FIG. 22A using CLIP and a resin
containing 87.1 wt % acrylic acid monomer, 2.52 wt % DPO photo
initiator, 10.4 wt % acetic acid, 0.17 wt % human insulin. Resin of
an identical composition, excluding the 1.75 wt % insulin, was used
as a negative control for insulin incorporation and release. Parts
were fabricated using continuous printing on the CLIP7 apparatus
(Carbon 3D) with a drawspeed of 100 mm/hr and 14.5 mW/cm2 light
intensity. CLIP fabricated PAA parts were dissolved in 2 mL of
water and the solution was then centrifuged at 20,000.times.g for
10 minutes to remove any insoluble material and insulin release was
quantified using an sandwich ELISA method (3A6 anti-insulin coating
antibody, 8E2-HRP detection antibody from AbCam) combined with and
an insulin standard curve. Insulin was readily detected in
dissolved CLIP fabricated PAA cylinders made with insulin
containing resin (FIG. 25) with an average of 0.5 unit/mL insulin
(1 unit/cylinder), which was close to the theoretical insulin
loading levels of 1 unit per cylinder based on PAA cylinder dry
weight (20-22 mg). No insulin was detected in the supernatant of
dissolved vehicle control cylinders (FIG. 25), validating the
specificity of the ELISA-based insulin assay. Without wishing to be
bound by theory, these results indicate that small peptide
pharmaceuticals have the capacity to be efficiently incorporated
and released from CLIP fabricated biomaterials.
[0216] Referring to FIG. 25, PAA cylinders were made with resin
containing 10.4 wt % acetic acid (Vehicle Control) or 50 units/mL
insulin in acetic acid (Insulin). Cylinders were dissolved in 2 mL
of water and the supernatant was tested for insulin release by
ELISA with values based on a standard curve.
[0217] d. Incorporation and Release of Model Protein from Clip
Microneedles
[0218] In order to demonstrate that CLIP microneedles are capable
of incorporating and releasing cargo, 0.1 wt % FITC-BSA
(Invitrogen) was mixed with PEG550-dMa (Sigma) at 97.4 wt % with
2.5 wt % DPO. This resin was used to make PEG microneedles on the
CLIP7 apparatus by exposing the resin to light at 1.5 mW/cm.sup.2
at 100 mm/hr. FITC-BSA was found to be evenly distributed through
the microneedle matrix via confocal microscopy (FIG. 26A). Release
of the FITC-BSA from these hydrogel needles in phosphate buffered
saline over 24 hours is shown in FIG. 26B. Without wishing to be
bound by theory, these results demonstrate that photopolymerizable
PEG microneedles fabricated using CLIP are capable of incorporating
and releasing proteins.
[0219] 6. Multi-Component Microneedles Produced by Mid-Production
Resin Exchange
[0220] An example of a multi-component microneedle produced using
CLIP is given in FIG. 27. In this example, the microneedle base is
composed of trimethylpropylol triacrylate whereas the tips are
composed of trimethylpropylol triacrylate plus rhodamine.
[0221] 7. Undercut Microneedles
[0222] Some arrowhead microneedles produced using CLIP are shown in
FIG. 28A. These microneedles were fabricated from a mixture of
TMPTA and 2.5 wt % DPO plus 0.1 wt % Mayzo BLS1326 ultraviolet
light absorber with 5.4 mW/cm.sup.2 of light at 41 mm/hr.
Microneedle dimensions are given in FIG. 28B. The specific
dimensions may be altered to optimize microneedle efficacy. Without
wishing to be bound by theory, it is hypothesized that these
microneedles will insert more deeply and more consistently into the
skin than a control microneedle, which contains no undercut. A
variety of other undercut microneedle shapes that could be produced
using CLIP are shown in FIG. 28C. The mechanical stability of such
shapes as compared to their efficacy in reducing microneedle
relaxation out of the skin is an area of potential study which
would allow for down-selection of ideal microneedle shapes.
[0223] 8. Tiered Microneedles
[0224] A tiered microneedle array was generated using CLIP
technology (FIG. 29). This array of TMPTA microneedles was
fabricated using equivalent techniques to those used in FIG. 19A-D.
Conical microneedles measure 300 .mu.m in diameter and 300, 600,
and 900 .mu.m in height.
[0225] Note that in addition to decreasing insertion forces, this
technique also circumvents the optimization problem that exists,
wherein increasing the available volume for drug loading in a
traditional array typically increases the force required for
insertion (FIG. 30). By using a tiered microneedle array, a larger
volume of therapeutic can be delivered using an equivalent force
(Table 4). These calculations assume that all tiers have the same
number of microneedles and that the base width of all microneedle
heights is equivalent.
TABLE-US-00004 TABLE 4 Force Volume of Required to # of MNs Cargo
Insert inserted inserted # of Equivalent # with MN Heights with
Tiers of MNs force F (.mu.M) force F 1 F X 1000 V 2 F/2 2X 1000,
900 1.9 V 3 F/3 3X 1000, 900, 800 2.7 V 4 F/4 4X 1000, 900, 800,
3.4 V 700 5 F/5 5X 1000, 900, 800, 4 V 700, 600 6 F/6 6X 1000, 900,
800, 4.5 V 700, 600, 500
[0226] 9. Microneedles With Curved or Discontinuous Sidewall
Profiles
[0227] Examples of microneedles fabricated with a curved and
discontinuous sidewall profile are shown in FIG. 5B. These
microneedles, which measure 1000 .mu.m in height and 500 .mu.m in
width, were fabricated using the CLIP Mini Apparatus (see Table 1)
at 100 mm/hr using 5.9 mW/cm.sup.2 of light. In various aspects, it
may be desirable to fabricate such microneedles out of two distinct
compositions, such as, for example, a strong material at the tip,
chosen for its material properties, over a soft base, chosen for
its chemical or biological properties, as shown in FIG. 5C. A
number of different specific conformations could be chosen, such as
those depicted in FIG. 5D.
[0228] 10. Fabrication of Microneedles with Water Soluble Chemical
Perforations
[0229] MNs were made using a computer-aided design (CAD) file
consisting of a needle patch backing, a needle shaft, and an
undercut arrowhead needle tip. The CAD file was converted to an STL
and sliced into 1 .mu.m cross sections using the Slic3r software to
create an SVG file. The backing and lower portion of the shaft
(slices 1-2750) were printed continuously using TMPTA+2 wt % TPO at
a speed of 100 mm/hour and light intensity of 5.8 mW/cm.sup.2. The
part was then washed of any residual resin using isopropanol while
affixed to the build platform. The resin in the reservoir was
exchanged for a water soluble resin consisting of acrylic acid
monomer +2 wt % DPO+1 wt % N-acetyl cysteine (NAC)+4.2 wt % water.
The NAC serves as a chain transfer agent to reduce the average
molecular weight of PAA chains to form a water soluble perforation.
Layers 2751-3250 were continuously printed using the water soluble
resin at a speed of 25 mm/hr with a light intensity of 9
mW/cm.sup.2. The part was then washed of any residual resin using
isopropanol while still affixed to the build platform. The resin in
the reservoir was exchanged for TMPTA+2 wt % TPO+0.01 wt %
Rhodamine, which was used to continuously print the undercut arrow
head structure (layers 3251-5368) at a speed of 100 mm/hour and
light intensity of 5.8 mW/cm.sup.2. The part was then rinsed a
final time with isopropanol and post-cured for 90 seconds under a
mercury lamp in the presence of nitrogen gas. The tri-material CLIP
fabricated needle containing a dissolvable perforation can be seen
(FIG. 31A, left). A control needle was made using an identical
process a loosely crosslinked acrylic acid resin containing a small
amount of the crosslinker TMPTA, which rendered the PAA water
insoluble. The base and lower shaft are made of TMPTA+2 wt % TPO,
the dissolvable layer is made of poly acrylic acid (PAA)+1 wt %
NAC. The non-dissolvable layer in the control needle is comprised
of PAA with 1 wt % water as a control. The tips for both needles
are made of non-dissolvable TMPTA+0.01 wt % rhodamine to serve as a
drug surrogate.
[0230] Both needles were submerged in water for 12 min., at which
time the dissolvable perforation dissolved and gave a clean
separation of the needle arrow head from the base/shaft (FIG. 31B,
left). In contrast the non-dissolvable perforation swelled and
maintained continuity between the needle shaft and arrowhead tip
(FIG. 31B, right).
[0231] The above method for CLIP fabrication of a needle structure
containing a dissolvable perforation utilizes the FDA approved
molecule N-Acetyl-Cysteine as a chain transfer agent to reduce the
molecular weight of PAA chains and facilitate solubility. Likewise,
other chain transfer agents, including other thiol containing
compounds (e.g. N-acetyl-cysteine, cysteine, DTT, 2-ME) and
non-thiol containing compounds (e.g. isopropanol, ethanol) could
also be added to resins for the purpose of controlling polymer
molecular weight and improve water solubility of parts fabricated
using CLIP technology.
[0232] 11. Fabrication of Dual Material Needles with Dissolvable
Tips and Non-Dissolvable Backing
[0233] CAD software was used to generate an STL file of a
microneedle patch containing 81 pyramidal needles with dimensions
of 1.2 mm in height and an aspect ratio of 3:1. The STL version of
this file was sliced into 1 .mu.m cross sections using the Slic3r
software and uploaded into the CLIP control panel as an SVG file.
Slices 1-900 encoded the 500 .mu.m base and 400 .mu.m of the 1.2 mm
tall needles, and were printed continuously using a non-dissolvable
hydrogel resin (poly(ethylene glycol)-diacrylate+2 wt % TPO) at 60
mm/hr with a light intensity of 2 mW/cm.sup.2. The part was then
washed of any residual resin using isopropanol and was post-cured
for 60 seconds under a mercury lamp in the presence of nitrogen
gas. The resin in the reservoir was exchanged for an acrylic acid
resin containing 0.7 wt % NAC+3 wt % H.sub.2O+2 wt % DPO+0.01 wt %
Rhodamine (as drug surrogate). This resin was used to make the
dissolvable microneedle tips by showing frames 801-1674 of the
sliced STL file with light intensity of 12 mW/cm.sup.2 at 25 mm/hr.
The needle patch was washed thoroughly with IPA and then post-cured
for 90 seconds under a mercury lamp in the presence of nitrogen
gas. The final microneedle patch is shown in FIG. 32. A control
microneedle patch was made using an identical process with the
omission of NAC from the acrylic acid resin, which renders the tip
water insoluble (FIG. 32).
[0234] These needles were applied to porcine skin ex vivo using
even pressure applied by gentle force with a 200 gram weight for 10
seconds. The weight remained in place on the skin for five minutes
and then the patch was removed and imaged. The tips on the needle
patch containing 0.7 wt % NAC dissolved completely after
application to the skin and the rhodamine dye is visible in the
porcine skin in a pattern that reflects the needle arrangement. In
contrast, the control needles lacking NAC swelled and bent after
application to the skin, but they did not dissolve. There was
little evidence of rhodamine deposition by the control needles in
the porcine skin.
[0235] The foregoing is illustrative of the present invention, and
is not to be construed as limiting thereof. The invention is
defined by the following claims, with equivalents of the claims to
be included therein.
[0236] It will be apparent to those skilled in the art that various
modifications and variations can be made in the present invention
without departing from the scope or spirit of the invention. Other
aspects of the invention will be apparent to those skilled in the
art from consideration of the specification and practice of the
invention disclosed herein. It is intended that the specification
and examples be considered as exemplary only, with a true scope and
spirit of the invention being indicated by the following claims
* * * * *