U.S. patent application number 14/458050 was filed with the patent office on 2014-11-27 for dissolvable microneedle arrays for transdermal delivery to human skin.
This patent application is currently assigned to University of Pittsburgh - Of the Commonwealth System of Higher Education. The applicant listed for this patent is Carnegie Mellon University, University of Pittsburgh - Of the Commonwealth System of Higher Education. Invention is credited to Geza Erdos, Louis D. Falo, JR., O. Burak Ozdoganlar.
Application Number | 20140350472 14/458050 |
Document ID | / |
Family ID | 43899029 |
Filed Date | 2014-11-27 |
United States Patent
Application |
20140350472 |
Kind Code |
A1 |
Falo, JR.; Louis D. ; et
al. |
November 27, 2014 |
DISSOLVABLE MICRONEEDLE ARRAYS FOR TRANSDERMAL DELIVERY TO HUMAN
SKIN
Abstract
A method of forming a microneedle array can include forming a
sheet of material having a plurality of layers and micromilling the
sheet of material to form a microneedle array. At least one of the
plurality of layers can include a bioactive component, and the
microneedle array can include a base portion and plurality of
microneedles extending from the base portion.
Inventors: |
Falo, JR.; Louis D.;
(Wexford, PA) ; Erdos; Geza; (Wexford, PA)
; Ozdoganlar; O. Burak; (Sewickley, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
University of Pittsburgh - Of the Commonwealth System of Higher
Education
Carnegie Mellon University |
Pittsburgh
Pittsburgh |
PA
PA |
US
US |
|
|
Assignee: |
University of Pittsburgh - Of the
Commonwealth System of Higher Education
Carnegie Mellon University
|
Family ID: |
43899029 |
Appl. No.: |
14/458050 |
Filed: |
August 12, 2014 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12910516 |
Oct 22, 2010 |
8834423 |
|
|
14458050 |
|
|
|
|
61279623 |
Oct 23, 2009 |
|
|
|
Current U.S.
Class: |
604/173 ;
264/138; 409/131 |
Current CPC
Class: |
A61L 2300/604 20130101;
Y10T 409/303752 20150115; A61L 2400/06 20130101; B23C 3/00
20130101; B23C 2220/48 20130101; B23C 2226/00 20130101; A61M
37/0015 20130101; B23C 2215/00 20130101; A61L 31/148 20130101; A61M
2037/0053 20130101; A61L 31/16 20130101; A61L 31/06 20130101; A61L
2300/426 20130101; A61L 31/042 20130101 |
Class at
Publication: |
604/173 ;
264/138; 409/131 |
International
Class: |
A61L 31/14 20060101
A61L031/14; A61L 31/06 20060101 A61L031/06; A61L 31/16 20060101
A61L031/16; A61L 31/04 20060101 A61L031/04; A61M 37/00 20060101
A61M037/00; B23C 3/00 20060101 B23C003/00 |
Goverment Interests
ACKNOWLEDGMENT OF GOVERNMENT SUPPORT
[0002] This invention was made with government support under
AI06008 and AI076060 awarded by the National Institutes of Health.
The government has certain rights in the invention.
Claims
1. A method of forming a microneedle array, comprising: forming a
sheet of material having a plurality of layers, at least one of the
plurality of layers comprising a bioactive component; and
micromilling the sheet of material to form a microneedle array, the
microneedle array comprising a base portion and plurality of
microneedles extending from the base portion.
2. The method of claim 1, wherein the plurality of layers comprise
a dissoluble biocompatible material.
3. The method of claim 2, wherein the dissoluble biocompatible
material is carboxymethylcellulose.
4. The method of claim 3, wherein the act of forming a sheet of
material having a plurality of layers comprises: providing a layer
of a hydrogel of carboxymethylcellulose to create a base layer, the
base layer having a substantially uniform thickness; drying the
base layer until the base layer is substantially solid; providing
one or more active layers above the base layer, the one or more
active layers having a substantially uniform thickness and
comprising a hydrogel of carboxymethylcellulose and a bioactive
component; and drying the one or more active layers until the one
or more active layers are substantially solid.
5. The method of claim 1, wherein the act of micromilling the sheet
of material comprises: micromilling the sheet of material to form a
plurality of microneedles that are generally pyramidal in
shape.
6. The method of claim 1, wherein the act of micromilling the sheet
of material comprises: micromilling the sheet of material to form a
plurality of pillar microneedles, each pillar microneedle
comprising a generally pyramidal section at a top portion of the
microneedle and a generally rectangular section at a bottom portion
of the microneedle.
7. The method of claim 6, wherein the act of micromilling the sheet
of material further comprises: micromilling a fillet portion on
each microneedle, the fillet portion being located at the area
where the microneedle contacts the base portion of the microneedle
array.
8. The method of claim 1, wherein the act of micromilling the sheet
of material comprises: forming the microneedle array so that each
microneedle comprises a first cross-sectional dimension at a top
portion, a second cross-sectional area at a bottom portion, and a
third cross-sectional dimension at an intermediate portion, wherein
the intermediate portion is located between the top portion and the
bottom portion, and the third cross-sectional dimension is greater
than the first and second cross-sectional dimensions.
9. The method of claim 1, wherein the bioactive component comprises
two or more different bioactive components.
10. The method of claim 9, wherein the bioactive component
comprises at least one antigen and at least one adjuvant for a
vaccine application.
11. A microneedle array formed by the process of claim 1.
12. A method comprising: forming a sheet of material having a
plurality of layers, at least one of the plurality of layers
comprising a bioactive component; and removing portions from the
sheet of material until a microneedle array is formed having a base
portion and plurality of microneedles extending from the base
portion, wherein the removal of portions from the sheet array
comprises forming the microneedle array so that each microneedle
comprises a first cross-sectional dimension at a top portion, a
second cross-sectional area at a bottom portion, and a third
cross-sectional dimension at an intermediate portion, the
intermediate portion being located between the top portion and the
bottom portion, and the third cross-sectional dimension being
greater than the first and second cross-sectional dimensions.
13. The method of claim 12, wherein the act of removing portions
from the sheet of material comprises micromilling the sheet of
material to form the plurality of microneedles.
14. The method of claim 13, further comprising: micromachining a
fillet portion on each microneedle, the fillet portion being
located at the area where the microneedle contacts the base portion
of the microneedle array.
15. The method of claim 12, wherein the bottom portion tapers
inward on all sides to the second cross-sectional dimension.
16. The method of claim 15, wherein respective microneedles
generally taper from the intermediate portion to a point above the
intermediate portion and generally taper from the intermediate
portion to a smaller cross-sectional dimension adjacent an area
where the microneedle contacts the base portion.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a divisional of U.S. application Ser. No.
12/910,516, filed Oct. 22, 2010, which claims the benefit of U.S.
Provisional Application No. 61/279,623, which was filed on Oct. 23,
2009. The prior applications are incorporated by reference herein
in their entirety.
FIELD
[0003] The disclosure pertains to systems and methods for
transdermal drug delivery, and, in particular, to systems and
methods for making and using dissolvable microneedle arrays.
BACKGROUND
[0004] The remarkable physical barrier function of the skin poses a
significant challenge to transdermal drug delivery. To address this
challenge, a variety of microneedle-array based drug delivery
devices have been developed. For example, one conventional method
employs solid or hollow microneedles arrays with no active
component. Such microneedle arrays can pre-condition the skin by
piercing the stratum corneum and the upper layer of epidermis to
enhance percutaneous drug penetration prior to topical application
of a biologic-carrier or a traditional patch. This method has been
shown to significantly increase the skin's permeability; however,
this method provides only limited ability to control the dosage and
quantity of delivered drugs or vaccine.
[0005] Another conventional method uses solid microneedles that are
surface-coated with a drug. Although this method provides somewhat
better dosage control, it greatly limits the quantity of drug
delivered. This shortcoming has limited the widespread application
of this approach and precludes, for example, the simultaneous
delivery of optimal quantities of combinations of antigens and/or
adjuvant in vaccine applications.
[0006] Another conventional method involves using hollow
microneedles attached to a reservoir of biologics. The syringe
needle-type characteristics of these arrays can significantly
increase the speed and precision of delivery, as well as the
quantity of the delivered cargo. However, complex fabrication
procedures and specialized application settings limit the
applicability of such reservoir-based microneedle arrays.
[0007] Yet another conventional method involves using solid
microneedle arrays that are biodegradable and dissolvable. This
method combines the physical toughness of solid microneedles with
relatively high bioactive material capacity, while retaining
desired attributes of simple fabrication, storage and application.
Current fabrication approaches for dissolvable polymer-based
microneedles generally use microcasting processes. For example, a
primary mastermold is commonly produced using a combination of
complex lithographic and laser etching technologies. However,
lithographic and laser-based technologies are limited in the range
of geometric features they can create, and the materials to which
they can be applied. Also, these highly complex fabrication
technologies do not allow rapid or low cost fabrication of
mastermolds, which can be particularly useful for systematic
testing of the bio-effectiveness of various different microneedle
and array geometries.
[0008] Accordingly, although transdermal delivery of biologics
using microneedle-array based devices offers attractive theoretical
advantages over prevailing oral and needle-based drug delivery
methods, considerable practical limitations exist in the design,
fabrication, and testing associated with microneedle arrays
constructed using conventional processes.
SUMMARY
[0009] The systems and methods disclosed herein include cutaneous
delivery platforms based on dissolvable microneedle arrays that can
provide efficient, precise, and reproducible delivery of
biologically active molecules to human skin. The microneedle array
delivery platforms can be used to deliver a broad range of
bioactive components to a patient.
[0010] In one embodiment, novel fabrication processes are provided
for producing microneedle arrays by micromilling a mastermold,
forming a production mold, and spin-casting material (including
bioactive components) into the production mold to create a
microneedle array. Such processes are flexible and enable simple
and rapid low cost production with efficient scale-up potential.
Further, because microneedles in these arrays can be configured to
not penetrate to the depth of vascular or neural structures,
delivery to human skin of bioactive components can be substantially
painless and bloodless.
[0011] In one embodiment, a method of forming a mastermold for
fabricating a microneedle array is provided. The method can include
providing a sheet of material; and micromilling the sheet of
material to form a mastermold having a base portion, a plurality of
projections extending from the base portion, and a fillet portion
between each of the projections and the base portion.
[0012] In another embodiment, a microneedle array can be formed by
directly micromilling a block or sheet of material. The method
includes forming a sheet of material having a plurality of layers
and micromilling the sheet of material to form a microneedle array.
At least one of the plurality of layers has a bioactive component.
The microneedle array can have a base portion and plurality of
microneedles extending from the base portion.
[0013] In specific implementations, the plurality of layers can
include a dissoluble biocompatible material. The dissoluble
biocompatible material can be carboxymethylcellulose. In other
specific implementations, the act of forming a sheet of material
can include the acts of providing a layer of a hydrogel of
carboxymethylcellulose to create a base layer of a substantially
uniform thickness;
[0014] drying the base layer until the base layer is substantially
solid; providing one or more active layers above the base layer
with a substantially uniform thickness, a hydrogel of
carboxymethylcellulose, and a bioactive component; and drying the
one or more active layers until the one or more active layers are
substantially solid. In specific implementations, the act of
micromilling includes micromilling the sheet of material to form a
plurality of microneedles that are generally pyramidal in shape. In
other specific implementations, the act of micromilling includes
micromilling the sheet of material to form a plurality of pillar
microneedles. Each pillar microneedle can include a generally
pyramidal section at a top portion of the microneedle and a
generally rectangular section at a bottom portion of the
microneedle.
[0015] In other specific implementations, the act of micromilling
includes micromilling a fillet portion on each microneedle. The
fillet portion can be located at the area where the microneedle
contacts the base portion of the microneedle array. In other
specific implementations, the act of micromilling the sheet
comprises forming each microneedle so that it comprises a first
cross-sectional dimension at a top portion, a second
cross-sectional area at a bottom portion, and a third
cross-sectional dimension at an intermediate portion. The
intermediate portion is located between the top portion and the
bottom portion, and the third cross-sectional dimension is greater
than the first and second cross-sectional dimensions.
[0016] In other specific implementations, the bioactive component
can include at least two different bioactive components. The
bioactive component can comprise an antigen and an adjuvant for a
vaccine application.
[0017] In another embodiment, a dissolvable microneedle array can
include a base portion and a plurality of microneedles. Each
microneedle can include a first cross-sectional dimension at a top
portion, a second cross-sectional dimension at a bottom portion,
and a third cross-sectional dimension at an intermediate portion.
The intermediate portion is located between the top portion and the
bottom portion, and the third cross-sectional dimension is greater
than the first and second cross-sectional dimensions.
[0018] In specific implementations, the top portion can include a
bioactive component. In other specific embodiments, each
microneedle can generally taper to a point above the intermediate
portion and each microneedle can generally taper to a smaller
cross-sectional dimension below the intermediate portion.
[0019] In other specific implementations, each microneedle can
include a fillet portion located at the area where each microneedle
contacts the base portion. In other specific implementations, each
microneedle can include a plurality of layers of dissoluble
biocompatible material. The dissoluble biocompatible material can
be carboxymethylcellulose. In other specific implementations, the
bioactive component can include at least two different bioactive
components. The bioactive component can include an antigen and an
adjuvant for a vaccine application.
[0020] In some embodiments, the bioactive components can comprise
dissoluble materials or insoluble but dispersible materials. The
bioactive components can be natural or formulated macro, micro and
nano particulates. The bioactive components can also comprise
mixtures of two or more of dissoluble, dispersible insoluble
materials and natural and/or formulated macro, micro and nano
particulates.
[0021] The structural and manufacturing advantages described
herein, coupled with a final product that can be stable at room
temperature, and inexpensive to transport and store, produce a
microneedle array that can be used for broad and rapid clinical
deployment. Taken together, these features can provide an
affordable and clinically feasible cutaneous delivery technology
capable of delivering a range of therapeutic agents, such as
vaccines useful in the prevention, treatment, or control of a broad
range of human diseases.
[0022] In certain embodiments, the microneedle arrays described
herein can be used for immunization procedures. In specific
implementations, the flexible delivery platforms can be used to
efficiently and simultaneously deliver both antigens and adjuvants
to skin resident dendritic cells, enabling both targeted antigen
delivery and adjuvant engineering of the immune response using the
same delivery platform.
[0023] The foregoing and other objects, features, and advantages of
the disclosed embodiments will become more apparent from the
following detailed description, which proceeds with reference to
the accompanying figures.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1 illustrates a miniature precision-micromilling system
used for fabricating microneedle mastermolds.
[0025] FIG. 2A is an enlarged view of a work-surface area of the
micro-milling system.
[0026] FIG. 2B is a schematic representation of the milled
mastermold.
[0027] FIG. 3 is an SEM image of a micromilled mastermold with
pyramidal needles.
[0028] FIG. 4 is an SEM image of a pyramidal needle shown in FIG.
3.
[0029] FIG. 5 is an SEM image of a pyramidal production mold.
[0030] FIG. 6 is an SEM image of an enlarged segment of the
production mold, illustrating a pyramidal needle molding well in
the center of the image.
[0031] FIG. 7A is an SEM image of a plurality of pyramidal-type
molded microneedles.
[0032] FIG. 7B is an SEM image of a single pyramidal-type molded
microneedle.
[0033] FIG. 8 is an SEM image of a pillar type molded
microneedle.
[0034] FIG. 9 is a micrograph of pyramidal type molded
microneedles.
[0035] FIG. 10 is a micrograph of pillar type molded
microneedles.
[0036] FIG. 11 illustrates a test apparatus for performing failure
and piercing tests.
[0037] FIG. 12 illustrates force-displacement curves for pillar
type microneedles (left) and pyramidal type microneedles
(right).
[0038] FIG. 13 illustrates a finite elements model of microneedle
deflections for pillar type microneedles (left) and pyramidal type
microneedles (right).
[0039] FIG. 14 show various stereo micrographs of the penetration
of pyramidal (A, C, E) and pillar (B, D, F) type microneedles in
skin explants.
[0040] FIGS. 15A, 15B, and 15C illustrate the effectiveness of
microneedle arrays in penetrating skin explants.
[0041] FIGS. 16A and 16B illustrate in vivo delivery of
particulates to the skin draining lymph nodes of microneedle array
immunized mice.
[0042] FIG. 17 is a bar graph showing immunogenicity of microneedle
delivered model antigens.
[0043] FIG. 18 is a bar graph showing the stability of the active
cargo of CMC-microneedle arrays in storage.
[0044] FIGS. 19A and 19B show induction of apoptosis in epidermal
cells that have been delivered CYTOXAN.RTM. (cyclophosphamide)
through a microneedle array.
[0045] FIG. 20 illustrates various microneedle geometries that can
be formed using micromilled mastermolds or by direct micromilling
of a block of material.
[0046] FIG. 21 illustrates a microneedle geometry that can be
formed by direct micromilling of a block of material.
[0047] FIG. 22 is a stereo microscopic image of a direct-fabricated
solid CMC-microneedle array.
[0048] FIG. 23 is a stereo microscopic image of a portion of the
microneedle array of FIG. 22.
[0049] FIG. 24 is a schematic cross-sectional view of a
casting-mold assembly for creating a block or sheet of material for
direct micromilling.
[0050] FIG. 25 is a schematic cross-sectional view of a drying
apparatus that can be used to dry a block or sheet of material for
direct micromilling.
DETAILED DESCRIPTION
[0051] The following description is exemplary in nature and is not
intended to limit the scope, applicability, or configuration of the
disclosed embodiments in any way. Various changes to the described
embodiment may be made in the function and arrangement of the
elements described herein without departing from the scope of the
disclosure.
[0052] As used in this application and in the claims, the singular
forms "a," "an," and "the" include the plural forms unless the
context clearly dictates otherwise. Additionally, the term
"includes" means "comprises." As used herein, the term "biologic,"
"active component," or "bioactive material" refers to
pharmaceutically active agents, such as analgesic agents,
anesthetic agents, anti-asthmatic agents, antibiotics,
anti-depressant agents, anti-diabetic agents, anti-fungal agents,
anti-hypertensive agents, anti-inflammatory agents, anti-neoplastic
agents, anxiolytic agents, enzymatically active agents, nucleic
acid constructs, immunostimulating agents, immunosuppressive
agents, vaccines, and the like. The bioactive material can comprise
dissoluble materials, insoluble but dispersible materials, natural
or formulated macro, micro and nano particulates, and/or mixtures
of two or more of dissoluble, dispersible insoluble materials and
natural and/or formulated macro, micro and nano particulates.
[0053] As used herein, the term "pre-formed" means that a structure
or element is made, constructed, and/or formed into a particular
shape or configuration prior to use. Accordingly, the shape or
configuration of a pre-formed microneedle array is the shape or
configuration of that microneedle array prior to insertion of one
or more of the microneedles of the microneedle array into the
patient.
[0054] Although the operations of exemplary embodiments of the
disclosed method may be described in a particular, sequential order
for convenient presentation, it should be understood that disclosed
embodiments can encompass an order of operations other than the
particular, sequential order disclosed. For example, operations
described sequentially may in some cases be rearranged or performed
concurrently. Further, descriptions and disclosures provided in
association with one particular embodiment are not limited to that
embodiment, and may be applied to any embodiment disclosed.
[0055] Moreover, for the sake of simplicity, the attached figures
may not show the various ways (readily discernable, based on this
disclosure, by one of ordinary skill in the art) in which the
disclosed system, method, and apparatus can be used in combination
with other systems, methods, and apparatuses. Additionally, the
description sometimes uses terms such as "produce" and "provide" to
describe the disclosed method. These terms are high-level
abstractions of the actual operations that can be performed. The
actual operations that correspond to these terms can vary depending
on the particular implementation and are, based on this disclosure,
readily discernible by one of ordinary skill in the art.
[0056] Micromilled Master Molds and Spin-Molded Microneedle
Arrays
[0057] In a first embodiment, apparatuses and methods are described
for fabricating dissolvable microneedle arrays using master molds
formed by micromilling techniques. For example, microneedle arrays
can be fabricated based on a mastermold (positive) to production
mold (negative) to array (positive) methodology. In contrast to the
formation of conventional mastermolds used to create microneedle
arrays, micromilling technology can be used to generate various
micro-scale geometries on virtually any type of material, including
metal, polymer, and ceramic parts. Micromilled mastermolds of
various shapes and configurations can be effectively used to
generate multiple identical female production molds. The female
production molds can then be used to microcast various microneedle
arrays.
[0058] Direct micromilling of mastermolds can replace expensive,
complex and equipment-sensitive SU-8 based lithography or laser
etching techniques, which are conventionally used to create
mastermolds for dissolvable needle arrays. In addition, as
discussed below, micromilling can provide for the construction of
more complex mastermold features than can conventional lithography
and laser etching processes.
[0059] FIG. 1 illustrates an example of a precision-micromilling
system that can be used for fabricating a microneedle mastermold.
Mechanical micromilling uses micro-scale (for example, as small as
10 .mu.m) milling tools within precision computer controlled
miniature machine-tool platforms. The system can include a
microscope to view the surface of the workpiece that is being cut
by the micro-tool. The micro-tool can be rotated at ultra-high
speeds (200,000 rpm) to cut the workpiece to create the desired
shapes.
[0060] As noted above, the micromilling process can be used to
create complex geometric features with many kinds of material,
which are not possible using conventional lithographic or laser
etching processes. Various types of tooling can be used in the
micromilling process, including, for example, carbide micro-tools.
In a preferred embodiment, however, diamond tools can be used to
fabricate the microneedle arrays on the master mold. Diamond
tooling can be preferable over other types of tooling because it is
harder than conventional materials, such as carbide, and can
provide cleaner cuts on the surface of the workpiece.
[0061] FIG. 2A illustrates a portion of the precision-micromilling
system shown in FIG. 1. In particular, FIG. 2A shows a workpiece
that is being micromilled by a micro-tool. FIG. 2B shows a
schematic enlarged view of a completed micromilled workpiece (i.e.,
a directly micromilled mastermold). As discussed in more detail
below, the completed micromilled workpiece can be a mastermold that
has a plurality of pyramidal projections extending from a base
surface of the workpiece.
[0062] Mastermolds can be micromilled from various materials,
including, for example, CIRLEX.RTM. (DuPont, KAPTON.RTM.
polyimide), which is the mastermold material described in the
exemplary embodiment. Mastermolds can be used to fabricate flexible
production molds from a suitable material, such as SYLGARD.RTM. 184
(Dow Corning, silicone elastomer), which is the production material
described in the exemplary embodiment below. The mastermold is
desirably formed of a material that is capable of being reused so
that a single mastermold can be repeatedly used to fabricate a
large number of production molds. Similarly each production mold is
desirably able to fabricate multiple microneedle arrays.
[0063] Mastermolds can be created relatively quickly using
micromilling technology. For example, a mastermold that comprises a
10 mm.times.10 mm array with 100 microneedles can take less than a
couple of hours and, in some embodiments, less than about 30
minutes to micromill. Thus, a short ramp-up time enables rapid
fabrication of different geometries, which permits the rapid
development of microneedle arrays and also facilitates the
experimentation and study of various microneedle parameters.
[0064] The mastermold material preferably is able to be cleanly
separated from the production mold material and preferably is able
to withstand any heighted curing temperatures that may be necessary
to cure the production mold material. For example, in an
illustrated embodiment, the silicone-based compound SYLGARD.RTM.
184 (Dow Corning) is the production mold material and that material
generally requires a curing temperature of about 80-90 degrees
Celsius.
[0065] Mastermolds can be created in various sizes. For example, in
an exemplary embodiment, a mastermold was created on 1.8 mm thick
CIRLEX.RTM. (DuPont, KAPTON.RTM. polyimide) and 5.0 mm thick
acrylic sheets. Each sheet can be flattened first by micromilling
tools, and the location where the microneedles are to be created
can be raised from the rest of the surface. Micro-tools can be used
in conjunction with a numerically controlled micromilling machine
(FIG. 1) to create the microneedle features (e.g., as defined by
the mastermold). In that manner, the micromilling process can
provide full control of the dimensions, sharpness, and spatial
distribution of the microneedles.
[0066] As shown in FIG. 3, a circular groove can be formed around
the microneedle array of the mastermold to produce an annular (for
example, circular) wall section in the production mold. The
circular wall section (FIG. 5) of the production mold can
facilitate the spincasting processes discussed below.
[0067] FIG. 3 is an image from a scanning electron microscope (SEM)
showing the structure of a micromilled mastermold with a plurality
of pyramidal needles. FIG. 4 illustrates an enlarged view of a
pyramidal needle of FIG. 3.
[0068] As discussed above, the production molds can be made from
SYLGARD.RTM. 184 (Dow Corning, silicone elastomer), which is a two
component clear curable silicone elastomer that can be mixed at a
10:1 SYLGARD.RTM. (Dow Corning, silicone elastomer) to curing agent
ratio. The mixture can be degassed for about 10 minutes and poured
over the mastermold to form an approximately 8 mm layer,
subsequently degassed again for about 30 minutes and cured at
85.degree. C. for 45 minutes. After cooling down to room
temperature, the mastermold can be separated from the cured
silicone, and the silicone production mold trimmed to the edge of
the circular wall section that surrounds the array (FIG. 5.). From
a single mastermold, a large number of production molds (e.g., 100
or more) can be produced with very little, if any, apparent
deterioration of the CIRLEX.RTM. (DuPont, KAPTON.RTM. polyimide) or
acrylic mastermolds.
[0069] FIG. 5 is an SEM image of a pyramidal production mold
created as described above. FIG. 6 illustrates an enlarged segment
of the production mold with a pyramidal needle molding well in the
center of the image. The molding well is configured to receive a
base material (and any components added to the base material) to
form microneedles with an external shape defined by the molding
well. To construct the microneedle arrays, a base material can be
used to form portions of each microneedle that have bioactive
components and portions that do not. Of course, if desired, each
microneedle can comprise only portions that contain bioactive
components; however, to control the delivery of the bioactive
component(s) and to control the cost of the microneedle arrays,
each microneedle preferably has a portion with a bioactive
component and a portion without a bioactive component.
[0070] Various materials can be used as the base material for the
microneedle arrays. The structural substrates of biodegradable
solid microneedles most commonly include poly(lactic-co-glycolic
acid) (PLGA) or carboxymethylcellulose (CMC) based formulations;
however, other bases can be used.
[0071] CMC is generally preferable to PLGA as the base material of
the microneedle arrays described herein. The PLGA based devices can
limit drug delivery and vaccine applications due to the relatively
high temperature (e.g., 135 degrees Celsius or higher) and vacuum
required for fabrication. In contrast, a CMC-based matrix can be
formed at room temperature in a simple spin-casting and drying
process, making CMC-microneedle arrays more desirable for
incorporation of sensitive biologics, peptides, proteins, nucleic
acids, and other various bioactive components.
[0072] CMC-hydrogel can be prepared from low viscosity sodium salt
of CMC with or without active components (as described below) in
sterile dH.sub.2O. In the exemplary embodiment, CMC can be mixed
with sterile distilled water (dH.sub.2O) and with the active
components to achieve about 25 wt % CMC concentration. The
resulting mixture can be stirred to homogeneity and equilibrated at
about 4 degrees Celsius for 24 hours. During this period, the CMC
and any other components can be hydrated and a hydrogel can be
formed. The hydrogel can be degassed in a vacuum for about an hour
and centrifuged at about 20,000 g for an hour to remove residual
micro-sized air bubbles that might interfere with a
spincasting/drying process of the CMC-microneedle arrays. The dry
matter content of the hydrogel can be tested by drying a fraction
(10 g) of it at 85 degrees Celsius for about 72 hours. The
ready-to-use CMC-hydrogel is desirably stored at about 4 degrees
Celsius until use.
[0073] Active components can be incorporated in a hydrogel of CMC
at a relatively high (20-30%) CMC-dry biologics weight ratio before
the spin-casting process. Arrays can be spin-cast at room
temperature, making the process compatible with the functional
stability of a structurally broad range of bioactive components.
Since the master and production molds can be reusable for a large
number of fabrication cycles, the fabrication costs can be greatly
reduced. The resulting dehydrated CMC-microneedle arrays are
generally stable at room temperature or slightly lower temperatures
(such as about 4 degrees Celsius), and preserve the activity of the
incorporated biologics, facilitating easy, low cost storage and
distribution.
[0074] In an exemplary embodiment, the surface of the production
molds can be covered with about 50 .mu.l (for molds with 11 mm
diameter) of CMC-hydrogel and spin-casted by centrifugation at
2,500 g for about 5 minutes. After the initial CMC-hydrogel layer,
another 50 .mu.l CMC-hydrogel can be layered over the mold and
centrifuged for about 4 hours at 2,500 g. At the end of a drying
process, the CMC-microneedle arrays can be separated from the
molds, trimmed off from excess material at the edges, collected and
stored at about 4 degrees Celsius. The production molds can be
cleaned and reused for further casting of microneedle arrays.
[0075] FIGS. 7A and 7B are SEM images of a CMC-microneedle array
formed with a plurality of pyramidal projections (i.e.,
microneedles). The average tip diameter of the pyramidal needles
shown in FIG. 7A is about 5-10 .mu.m. As shown in FIG. 7B, the
sides of the pyramidal needles can be formed with curved and/or
arcuate faces that can facilitate insertion in skin.
[0076] FIG. 8 is another SEM image of a single needle of a
microneedle array. The microneedle shown in FIG. 8 is a
base-extended pillar type molded CMC-microneedle. The base-extended
pillar type microneedle comprises a base portion, which is
generally polyagonal (for example, rectangular) in cross section,
and a projecting portion that extends from the base portion. The
projecting portion has a lower portion that is substantially
rectangular and tip portion that generally tapers to a point. The
tip portion is generally pyramidal in shape, and the exposed faces
of the pyramid can be either flat or arcuate. The projecting
portion can be half or more the entire length of the needle.
[0077] FIGS. 9 and 10 illustrate micrographs of pyramidal (FIG. 9)
and pillar type (FIG. 10) molded CMC-microneedles. Because the
pyramidal needles have a continually increasing cross-sectional
profile (dimension) from the needle point to the needle base, as
the needle enters the skin, the force required to continue pushing
the pyramidal needle into the skin increases. In contrast, pillar
type needles have a generally continuous cross-sectional profile
(dimension) once the generally rectangular portion of the
projection portion is reached. Thus, pillar type needles can be
preferable over pyramidal type needles because they can allow for
the introduction of the needle into the skin with less force.
[0078] FIG. 20 illustrates schematic representation of microneedle
shapes and structures that are generally suitable for fabrication
by spin-casting material into a mastermold formed by micromilling.
Since the shapes and structures shown in FIG. 20 do not contain any
undercuts, they generally will not interfere with the
molding/de-molding process. The structures in FIG. 20 include (a) a
generally pyramidal microneedle, (b) a "sharp" pillar type
microneedle (without the base member of FIG. 8), (c) a "wide"
pillar type microneedle, (d) a "short" pillar type microneedle
(having a short pillar section and a longer pointed section), and
(e) a "filleted" pillar type microneedle.
[0079] While the volume of the pyramidal microneedles can be
greater than that of the pillar type microneedles, their increasing
cross-sectional profile (dimension) requires an increasing
insertion force. Accordingly, the geometry of the pyramidal
microneedles can result in reduced insertion depths and a reduced
effective delivery volume. On the other hand, the smaller
cross-sectional area and larger aspect ratio of the pillar
microneedles may cause the failure force limit to be lower. The
smaller the apex angle .alpha., the "sharper" the tip of the
microneedle. However, by making the apex angle too small (e.g.,
below about 30 degrees), the resulting microneedle volume and
mechanical strength may be reduced to an undesirable level.
[0080] The penetration force of a microneedle is inversely
proportional to the microneedle sharpness, which is characterized
not only by the included (apex) angle of the microneedles, but also
by the radius of the microneedle tip. While the apex angle is
prescribed by the mastermold geometry, the tip sharpness also
depends on the reliability of the mold. Micromilling of mastermolds
as described herein allows for increased accuracy in mold geometry
which, in turn, results in an increased accuracy and reliability in
the resulting production mold and the microneedle array formed by
the production mold.
[0081] The increased accuracy of micromilling permits more accurate
and detailed elements to be included in the mold design. For
example, as discussed in the next section below, the formation of a
fillet at the base of a pillar type microneedle can significantly
increase the structural integrity of the microneedle, which reduces
the likelihood that the microneedle will fail or break when it
impacts the skin. While these fillets can significantly increase
the strength of the microneedles, they do not interfere with the
functional requirements of the microneedles (e.g., penetration
depth and biologics volume). Such fillets are very small features
that can be difficult to create in a master mold formed by
conventional techniques. However, the micromilling techniques
described above permit the inclusion of such small features with
little or no difficulty.
[0082] Mechanical Integrity and Penetration Capabilities
[0083] Microneedle arrays are preferably configured to penetrate
the stratum corneum to deliver their cargo (e.g., biologics or
bioactive components) to the epidermis and/or dermis, while
minimizing pain and bleeding by preventing penetration to deeper
layers that may contain nerve endings and vessels. To assess the
mechanical viability of the fabricated microneedle arrays, tests
were performed on the pyramidal and pillar type microneedle arrays
as representative variants of array geometry (shown, e.g., in FIGS.
7B and 8). The first set of tests illustrate the failure limit of
microneedles, and include pressing the microneedle array against a
solid acrylic surface with a constant approach speed, while
simultaneously measuring the force and the displacement until
failure occurs. The second set of tests illustrate the piercing
capability of the microneedles on human skin explants.
[0084] FIG. 11 illustrates a test apparatus designed for functional
testing. The sample (i.e., microneedle array) was attached to a
fixture, which was advanced toward a stationary acrylic artifact
(PMMA surface) at a constant speed of about 10 mm/s speed using a
computer-controlled motion stage (ES14283-52 Aerotech, Inc.). A
tri-axial dynamometer (9256C1, Kistler, Inc.) that hosted the
acrylic artifact enabled high-sensitivity measurement of the
forces.
[0085] FIG. 12 illustrates force-displacement curves of data
measured during failure tests. The curve on the left is
representative of data obtained from testing a pillar microneedle
sample and the curve on the right is representative of data
obtained from testing a pyramid microneedle. As seen in FIG. 12,
the failure of these two kinds of microneedles are significantly
different; while the pyramidal arrays plastically deform (bend),
the pillar type arrays exhibit breakage of the pillars at their
base. This different failure behavior lends itself to considerably
different displacement-force data. The failure (breakage) event can
be easily identified from the displacement-force data as indicated
in the figure. Based on the obtained data, the failure point of
pillar type microneedles was seen to be 100 mN in average. As only
about 40 mN of force is required for penetration through the
stratum corneum, the microneedles are strong enough to penetrate
human skin without failure. Furthermore, since parallelism between
microneedle tips and the acrylic artifact cannot be established
perfectly, the actual failure limit will likely be significantly
higher than 100 mN (i.e., microneedles broke in a successive
manner, rather than simultaneous breakage of most/all
microneedles).
[0086] The pyramidal microneedles presented a continuously
increasing force signature with no clear indication of point of
failure. To identify the failure limit for the pyramidal
microneedles, interrupted tests were conducted in which the
microneedles were advanced into the artifact by a certain amount,
and retreated and examined through optical microscope images. This
process was continued until failure was observed. For this purpose,
the failure was defined as the bending of the pyramidal
microneedles beyond 15 degrees.
[0087] To further analyze the failure of the microneedles, the
finite-elements model (FEM) of the microneedle arrays shown in FIG.
13 was developed. To obtain the mechanical properties (elastic
modulus and strength limit) of the CMC material, a series of
nanoindentation tests (using a Hysitron nanoindentor). The average
elastic modulus and yield strength of the CMC material (as
prepared) were 10.8 GPa and 173 MPa, respectively. This indicates
that the prepared CMC material has a higher elastic modulus and
yield strength than both PMMA (elastic modulus: 3.1 GPa, yield
strength: 103 MPa) and polycarbonate (elastic modulus: 2.2 GPa,
yield strength: 75 MPa), indicating the superior strength and
stiffness of CMC material with respect to other polymers.
[0088] Using this data, a series of FEM simulations were conducted.
It was predicted from the FEM models that failure limit of
pyramidal and sharp-pillar (width=134 .mu.m) microneedles with 600
.mu.m height, 30 degree apex angle, and 20 .mu.m fillet radius were
400 mN (pyramid) and 290 mN (sharp-pillar) for asymmetric loading
(5 degrees loading misorientation). Considering that the minimum
piercing force requirement is about 40 mN, pyramid and sharp-pillar
microneedles would have factors of safety of about 10 and 7.25,
respectively.
[0089] When the fillet radius is doubled to 40 .mu.m, the failure
load for the pillar was increased to 350 mN, and when the fillet
radius is reduced to 5 .mu.m, the failure load was reduced to 160
mN, which is close to the experimentally determined failure load.
The height and width of the pillars had a significant effect on
failure load. For instance, for 100 .mu.m width pillars, increasing
the height from 500 .mu.m to 1000 .mu.m reduced the failure load
from 230 mN to 150 mN. When the width is reduced to 75 .mu.m, for a
750 .mu.m high pillar, the failure load was seen to be 87 mN.
[0090] To evaluate penetration capability, pyramidal and
sharp-pillar microneedle arrays were tested for piercing on
water-based model elastic substrates and on full thickness human
skin. FIG. 14 illustrates stereo micrographs of pyramidal (Panels
A, C, and E) and pillar type microneedle arrays (B, D, and F) after
4 minutes of exposure to model elastics. In particular, toluene
blue tracer dye was deposited in model elastic substrates (Panels C
and D) or freshly excised full thickness human skin explants
(Panels E and F) after application of pyramidal or pillar type
microneedle arrays.
[0091] The model elastic substrate comprised about 10% CMC and
about 10% porcine gelatin in PBS gelled at about 4 degrees Celsius
for about 24 hours or longer. The surface of the elastics was
covered with about 100 .mu.m thick parafilm to prevent the
immediate contact of the needle-tips and the patch materials with
the water based model elastics. To enable stereo
microscopic-imaging, trypan blue tracer dye (Sigma Chem., cat
#T6146) was incorporated into the CMC-hydrogel at 0.1%
concentration. The patches were applied using a spring-loaded
applicator and analyzed after about a 4 minute exposure. Based on
physical observation of the dye in the target substrates, the
dissolution of the microneedles of the two different geometries was
markedly different.
[0092] The sharp-pillar needles applied to the model elastic
substrate released substantially more tracer dye to the gel matrix
than that observed for the pyramidal design (FIG. 14, C vs. D).
Images of the recovered patches (FIG. 14, A vs. B) were consistent
with this observation, as the degradation of the sharp-pillar
needles was more advanced than that of the pyramidal needles. To
extrapolate this analysis to a more clinically relevant model,
pyramidal and pillar type microneedle arrays were applied to
freshly excised full thickness human skin explants using the same
force from the spring loaded applicator. Consistent with results
from the elastic model, the pyramidal microneedle arrays deposited
visibly less tracer dye than the sharp-pillar microneedle arrays
(FIG. 14, E vs. F).
[0093] To further evaluate penetration and to assess delivery
effectiveness to human skin, CMC-microneedle arrays were fabricated
with BIOMAG.RTM. (Polysciences, Inc., cat#. 84100) beads or
fluorescent particulate tracers (FLURESBRITE.RTM., YG 1 .mu.m,
Polysciences Inc., cat#. 15702). The pyramidal CMC-microneedle
arrays containing fluorescent or solid particulates were applied to
living human skin explants as described previously. Five minutes
after the application, surface residues were removed and skin
samples were cryo-sectioned and then counterstained with toluene
blue for imaging by light microscopy (FIGS. 15A and 15B) or by
fluorescent microscopy (FIG. 15C).
[0094] Pyramidal CMC-microneedles effectively penetrated the
stratum corneum, epidermis, and dermis of living human skin
explants, as evidenced by the deposition of Biomag beads lining
penetration cavities corresponding to individual needle insertion
points (representative sections shown in FIGS. 15A and 15B). In
particular, ordered cavities (FIG. 15A, cavities numbered 1-4,
toluene blue counterstain, 10.times.) and deposits of BioMag
particles (brown) lining penetration cavities were evident (FIG.
15B, 40.times.), indicating microneedle penetrated of human skin.
Further, analysis of sections from living human explants stained
with DAPI to identify cell nuclei and anti-HLA-DR to identify MHC
class II+ antigen presenting cells revealed high density
fluorescent particulates deposited in the superficial epidermis and
dermis, including several particles co-localized with class II+
antigen presenting cells (FIG. 15C, DAPI (blue), HLA-DR+ (red) and
fluorescent particles (green), 40.times.).
[0095] These results further demonstrate that the CMC microneedle
arrays described herein can effectively penetrate human skin and
deliver integral cargo (bioactive components), including insoluble
particulates. They are consistent with effective delivery of
particulate antigens to antigen presenting cells in human skin,
currently a major goal of rational vaccine design.
[0096] To further address microneedle array delivery in vivo, the
cutaneous delivery of particulate antigen in vivo was modeled by
similarly applying fluorescent particle containing arrays to the
dorsal aspect of the ears of anesthetized mice. After 5 minutes,
patches were removed and mice resumed their normal activity. Three
hours or 3 days, ear skin and draining lymph nodes were analyzed
for the presence of fluorescent particles. Consistent with
observations of human skin, particulates were evident in the skin
excised from the array application site (data not shown). Further,
at the 3 day time point, substantial numbers of particles were
evident in the draining lymph nodes. FIGS. 16A and 16B illustrates
substantial numbers of particles that were evident in the draining
lymph Nodes (FIG. 16A, 10.times.), including clusters of
particulates closely associated with Class II+ cells (FIG. 16B,
60.times.) suggesting the presence of lymph node resident antigen
presenting cells with internalized particulates.
[0097] To quantitatively evaluate the effects of needle geometry on
cargo delivery using microneedle arrays, 3H-tracer labeled
CMC-microneedle arrays were constructed. The CMC-hydrogel was
prepared with 5% wt ovalbumin as a model active component at 25 wt
% final dry weight content (5 g/95 g OVA/CMC) and trace labeled
with 0.1 wt % trypan blue and 0.5.times.10.sup.6 dpm/mg dry weight
3H-tracer in the form of 3H-thymidine (ICN Inc., cat #2406005).
From a single batch of labeled CMC-hydrogel-preparation four
batches of 3H-CMC-microneedle arrays were fabricated, containing
several individual patches of pyramidal and sharp-pillar needle
geometry. The patches were applied to human skin explants as
described above and removed after 30 min exposure. The
patch-treated area was tape-striped to remove surface debris and
cut using a 10 mm biopsy punch. The 3H content of the excised human
skin explants-discs was determined by scintillation counting. The
specific activity of the 3H-CMC-microneedle patch-material was
determined and calculated to be 72,372 cpm/mg dry weight. This
specific activity was used to indirectly determine the amount of
ovalbumin delivered to and retained in the skin. The resulting data
is summarized in Table 1 below.
[0098] The tested types of patches were consistent from microneedle
array to microneedle array (average standard deviation 24-35%) and
batch to batch (average standard deviation 7-19%). The intra-batch
variability for both needle geometry was lower than the in-batch
value indicating that the insertion process and the characteristics
of the target likely plays a primary role in the successful
transdermal material delivery and retention. The patch-material
retention data clearly demonstrate the foremost importance of the
microneedle geometry in transdermal cargo delivery. Pillar-type
needle geometry afforded an overall 3.89 fold greater deposition of
the 3H labeled needle material than that of the pyramidal needles.
On the basis of the deposited radioactive material, it is estimated
that the pyramidal needles were inserted about 200 .mu.m deep while
the pillar-type were inserted about 400 .mu.m or more.
TABLE-US-00001 TABLE 4.2.5 Transfer of .sup.3H-labeled
CMC-microneedle material into human skin explants by pyramidal and
pillar-type needles. Pyramidal Pillar-Type Needles Needles Pyramid
OVA Pillar-Type OVA Pillar to Array Needles STDev Transferred
Needles STDev Transferred Pyramid Batches (cpm/patch) (%)
(.mu.g/patch) (cpm/patch) (%) (.mu.g/patch) Ratio Batch A 2459.00
17.56 1.70 11700.50 31.52 8.08 4.76 Batch B 3273.50 57.39 2.26
12816.50 21.45 8.85 3.92 Batch C 2757.75 46.13 1.90 12240.00 26.77
8.46 4.44 Batch D 3782.00 36.27 2.61 10921.50 9.32 7.55 2.89
IntraBatch 3088.06 19.00 2.12 11919.63 6.77 8.24 3.89 AVG
[0099] Desirably, the microneedle arrays described herein can be
used for cutaneous immunization. The development of strategies for
effective delivery of antigens and adjuvants is a major goal of
vaccine design, and immunization strategies targeting cutaneous
dendritic cells have various advantages over traditional
vaccines.
[0100] The microneedle arrays described herein can also be
effective in chemotherapy and immunochemotherapy applications.
Effective and specific delivery of chemotherapeutic agents to
tumors, including skin tumors is a major goal of modern tumor
therapy. However, systemic delivery of chemotherapeutic agents is
limited by multiple well-established toxicities. In the case of
cutaneous tumors, including skin derived tumors (such as basal
cell, squamous cell, Merkel cell, and melanomas) and tumors
metastatic to skin (such as breast cancer, melanoma), topical
delivery can be effective. 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.
[0101] The microneedle arrays of the present disclosure can be used
as an alternative to or in addition to traditional topical
chemotherapy approaches. The microneedle arrays of the present
disclosure can penetrate the outer layers of the skin and
effectively deliver the active biologic to living cells in the
dermis and epidermis. Delivery of a chemotherapeutic agents results
in the apoptosis and death of skin cells.
[0102] Further, multiple bioactive agents can be delivered in a
single microneedle array (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.
[0103] In an exemplary embodiment, the delivery of a biologically
active small molecule was studied. In particular, the activity of
the chemotherapeutic agent CYTOXAN.RTM. (cyclophosphamide)
delivered to the skin with CMC microneedle arrays was studied. The
use of CYTOXAN.RTM. (cyclophosphamide) enables direct measurement
of biologic activity (CYTOXAN.RTM. (cyclophosphamide) induced
apoptosis in the skin) with a representative of a class of agents
with potential clinical utility for the localized treatment of a
range of cutaneous malignancies.
[0104] To directly evaluate the immunogenicity of CMC microneedle
array incorporated antigens, the well characterized model antigen
ovalbumin was used. Pyramidal arrays were fabricated incorporating
either soluble ovalbumin (sOVA), particulate ovalbumin (pOVA), or
arrays containing both pOVA along with CpGs. The adjuvant effects
of CpGs are well characterized in animal models, and their
adjuvanticity in humans is currently being evaluated in clinical
trials.
[0105] Immunization was achieved by applying antigen containing
CMC-microneedle arrays to the ears of anesthetized mice using a
spring-loaded applicator as described above, followed by removal of
the arrays 5 minutes after application. These pyramidal microneedle
arrays contained about 5 wt % OVA in CMC and about 0.075 wt % (20
.mu.M) CpG. As a positive control, gene gun based genetic
immunization strategy using plasmid DNA encoding OVA was used. Gene
gun immunization is among the most potent and reproducible methods
for the induction of CTL mediated immune responses in murine
models, suggesting its use as a "gold standard" for comparison in
these assays.
[0106] Mice were immunized, boosted one week later, and then
assayed for OVA-specific CTL activity in vivo. Notably,
immunization with arrays containing small quantities of OVA and CpG
induced high levels of CTL activity, similar to those observed by
gene gun immunization (FIG. 17). Significant OVA-specific CTL
activity was elicited even in the absence of adjuvant, both with
particulate and soluble array delivered OVA antigen. It is well
established that similar responses require substantially higher
doses of antigen when delivered by traditional needle
injection.
[0107] To evaluate the stability of fabricated arrays, batches of
arrays were fabricated, stored, and then used over an extended
period of time. As shown in FIG. 18, no significant deterioration
of immunogenicity was observed over storage periods spanning up to
80 days (longest time point evaluated). Thus, the CMC microneedle
arrays and this delivery technology can enable effective cutaneous
delivery of antigen and adjuvants to elicit antigen specific
immunity.
[0108] To evaluate the delivery of a biologically active small
molecule, pyramidal CMC-microneedle arrays were fabricated with the
low molecular weight chemotherapeutic agent CYTOXAN.RTM.
(cyclophosphamide), or with FLURESBRITE.RTM. (Polysciences, Inc.)
green fluorescent particles as a control. CYTOXAN.RTM.
(cyclophosphamide) was integrated at a concentration of 5 mg/g of
CMC, enabling delivery of approximately about 140 .mu.g per array.
This is a therapeutically relevant concentration based on the area
of skin targeted, yet well below levels associated with systemic
toxicities. Living human skin organ cultures were used to assess
the cytotoxicty of CYTOXAN.RTM. (cyclophosphamide). CYTOXAN.RTM.
(cyclophosphamide) was delivered by application of arrays to skin
explants as we previously described. Arrays and residual material
were removed 5 minutes after application, and after 72 hours of
exposure, culture living skin explants were cryo-sectioned and
fixed. Apoptosis was evaluated using green fluorescent TUNEL assay
(In Situ Cell Death Detection Kit, TMR Green, Roche,
cat#:11-684-795-910). Fluorescent microscopic image analysis of the
human skin sections revealed extensive apoptosis of epidermal cells
in CYTOXAN.RTM. (cyclophosphamide) treated skin as shown in FIG.
19A. As shown in FIG. 19B, no visible apoptosis was observed in
fluorescent particle treated skin though these particles were
evident, validating that the observed area was accurately targeted
by the microneedle array.
[0109] Direct Fabricated Microneedle Arrays
[0110] The micromilling of mastermolds described above allows the
production of microneedle arrays with a variety of geometries. In
another embodiment, systems and methods are provided for
fabricating a microneedle array by directly micromilling various
materials, such as dried CMC sheets. The same general tooling that
was described above with respect to the micromilling of mastermolds
can be used to directly micromilling microneedle arrays.
[0111] Direct micromilling of microneedle arrays eliminates the
need for molding steps and enables a simplified, scalable, and
precisely reproducible production strategy that will be compatible
with large scale clinical use. Moreover, direct fabrication of the
microneedle arrays through micromilling enables greater control of
microneedle geometries. For example, micromilling permits the
inclusion of microneedle retaining features such as undercuts
and/or bevels, which cannot be achieved using molding
processes.
[0112] The reproducibility of direct milling of microneedle arrays
is particular beneficial. That is, in direct micromilling all of
the microneedles are identical as a result of the milling
fabrication process. In molding operations, it is not uncommon for
some needles to be missing or broken from a given patch as a result
of the process of physically separating them from the molds. For
use in certain medical applications, the reproducibility of the
amount of bioactive components in the array is very important to
provide an appropriate level of "quality control" over the process,
since irregularities in the needles from patch to patch would
likely result in variability in the dose of drug/vaccine delivered.
Of course, reproducibility will also be an important benefit to any
application that requires FDA approval. Spincast/molded patches
would require special processes to assure acceptable uniformity for
consistent drug delivery. This quality control would also be likely
to result in a certain percentage of the patches "failing" this
release test, introducing waste into the production process. Direct
micromilling eliminates or at least significantly reduces these
potential problems.
[0113] Molding processes also have inherent limitations because of
the need to be able to fill a well or concavity and remove the
cured molded part from that well or concavity. That is because of
mold geometries, undercuts must generally be avoided when molding
parts or the part will not be removable from the mold. That is, a
geometrical limitation of a molded part, such as a molded
microneedle array, is that any feature located closer to the apex
must be narrower than any feature located toward the base.
[0114] Accordingly, in view of these limitations, FIG. 20
illustrates schematic representation of microneedle shapes and
structures that are generally suitable for fabrication by molding.
That is, the shapes and structures shown in FIG. 20 do not contain
any undercuts that would prevent the part (i.e., the microneedles)
from being removed from a production mold. In contrast, FIG. 21
illustrates a beveled, undercut microneedle shape that cannot be
molded in the manners described herein. This geometry can only be
created through direct fabrication using the proposed micromilling
technology. The negative (bevel) angle facilitates better retention
of the microneedles in the tissue. In addition, because the
microneedle of FIG. 21 has a wider intermediate portion (with a
larger cross-sectional dimension) above a lower portion (with a
smaller cross-sectional dimension), a greater amount of the
bioactive material can be delivered by configuring the microneedle
to hold or store the bioactive material in the wider section, which
is configured to be retained within the skin. Thus, the larger
cross-sectional dimension of the intermediate portion can "carry"
the bulk of the bioactive component. Since the lower portion tapers
to a narrower cross-sectional dimension, the wider intermediate
portion will obtain good penetration for delivery of the bioactive
component into the skin layer. A portion above the intermediate
portion desirably narrows to a point to facilitate entry of the
microneedles into the skin layers.
[0115] Another limitation of molded parts is that it can be
difficult to precisely fill a very small section of a mold. Since
production molds for microneedle arrays comprise numerous very
small sections, it can be difficult to accurately fill each well.
This can be particularly problematic when the mold must be filled
with different materials, such as a material that contains a
bioactive component and a material that does not contain a
bioactive component. Thus, if the production mold is to be filled
with layers, it can be difficult to accurately fill the tiny wells
that are associated with each microneedle. Such reproducibility is
particularly important, since the microneedles are intended to
deliver one or more bioactive components. Thus, even slight
variations in the amounts of bioactive component used to fill
production molds can be very undesirable.
[0116] Also, by using a lamination structure to form a sheet or
block that can be micromilled, various active components can be
integrated into a single microneedle by vertical layering. For
example, in an exemplary embodiment, CMC-hydrogel and
CMC-sOVA-hydrogel (80% CMC/20 wt % OVA) were layered into the form
of a sheet or block. This composite sheet can be micro-machined
using the direct micromilling techniques described herein.
[0117] FIG. 19 is a stereo-microscopic image analysis of an entire
microneedle array. The microneedle comprises a 10.times.10 array of
microneedles. FIG. 20 is an enlarged segment of the microneedle
array of FIG. 19. The layering of two components is shown in FIG.
20, which illustrates darker areas of the microneedles at tip
portions and lighter areas of the microneedles at base portions.
The darker layer at the tip represents the layer comprising a
bioactive component, in this case soluble ovalbumin contained in a
CMC layer.
[0118] Although the formation of a layer containing active material
(e.g., antigen) and the subsequent micromilling of the layer (and
any other adjacent layers) may require the use of relatively large
amounts of the active material, the material can be removed (e.g.,
in the form of chips), recovered, and recycled. Direct machining
technology is not restricted by the geometrical constraints arising
from the molding/de-molding approach, and thus, is capable of
creating more innovative needle designs (e.g., FIG. 21), which can
significantly improve the retained needle-volume and needle
retention time in the skin.
[0119] The production of sheets or blocks by forming a plurality of
layers can provide a solid material that can be micro-machined and
which can comprise one or more layers with a bioactive component.
For example, a dissoluble solid carboxymethylcellulose polymer
based block or sheet with well-defined and controlled dimensions
can be fabricated by a lamination process. The resulting sheet or
block can be fully machineable, similar to the machining of plastic
or metal sheets or blocks. As described herein, the fabrication
process can be suitable for the incorporation of bioactive
components into the matrix without significantly reducing their
activity levels.
[0120] As described below, a fabricated sheet of material (such as
a CMC based material) can be directly micro-machined/micromilled)
to produce one or more microneedle arrays suitable for delivering
active ingredients through the skin. This dissoluble biocompatible
CMC block-material can be used for the delivery of soluble or
insoluble and particulate agents in a time release manner for body
surface application. The biocompatible material can be suitable for
implants in deeper soft or hard tissue when dissolution of the
scaffolding material is required and useful.
[0121] The following method can be used to prepare a
carboxymethylcellulose (CMC) polymer low viscosity hydrogel to
12.5% concentration. The 12.5% carboxymethylcellulose (CMC) low
viscosity hydrogel can be prepared in water or other biocompatible
buffer, such as (but not limited to) PBS or HBS. During the
preparation of the polymer solution, soluble agents (such as
nucleic acid, peptides, proteins, lipids or other organic and
inorganic biologically active components) and particulates can be
added (e.g. ovalbumin, a soluble agent). Ferrous particulates
carrying active ingredients at 20 w/w % of CMC can be used.
[0122] The preparation of 1000 g sterile 12.5% CMC hydrogel with no
active component can be achieved as follows:
[0123] 1) Measure 125 g CMC, add 875 g water or other water based
solvent.
[0124] 2) Stir to homogeneity in overhead mixer.
[0125] 3) Autoclave homogenate to sterility at 121 degrees Celsius
for 1 hour (the autoclaving step can reduce viscosity for improved
layering)
[0126] 4) Cool to 22 degrees Celsius.
[0127] 5) Vacuum treat the resulting material at 10 torr and 22
degrees Celsius for 1 hour to remove trapped micro-bubbles.
[0128] 6) Centrifuge product at 25,000 g for 1 hour in vacuum
chambered centrifuge (for floating and further removing residual
micro bubbles).
[0129] 7) Store the CMC-hydrogel product at 4 degrees Celsius.
[0130] The preparation of 1000 g sterile 12.5 w/w % dry content
20/80% ovalbumin/CMC hydrogel can be achieved as follows:
[0131] 1) Measure 100 g CMC add 650 g water or other water based
solvent.
[0132] 2) Stir to homogeneity in overhead mixer.
[0133] 3) Autoclave homogenate to sterility at 121 degrees Celsius
for 1 hour (this autoclaving step can reduce viscosity for improved
layering).
[0134] 4) Cool to 22 degrees Celsius.
[0135] 5a) Dissolve 25 g ovalbumin in 225 g water.
[0136] 5b) Sterile filter ovalbumin solution on 0.22 .mu.m pore
sized filter.
[0137] 6) Mix to homogeneity, under sterile conditions the 750 g
CMC hydrogel with 250 g sterile ovalbumin solution.
[0138] 7) Vacuum treat the resulting material at 10 torr and 22
degrees Celsius for 1 hour to remove trapped micro-bubbles.
[0139] 8) Centrifuge product at 25,000 g for 1 hour in vacuum
chambered centrifuge (for floating and further removing residual
micro bubbles).
[0140] 9) Store the CMC-hydrogel product at 4 degrees Celsius.
[0141] The preparation of 100 g sterile 12.5 w/w % dry content
20/80% particulate-ovalbumin/CMC hydrogel can be achieved as
follows:
[0142] 1) Measure 10 g CMC add 87.5 g water or other water based
solvent.
[0143] 2) Stir to homogeneity in overhead mixer.
[0144] 3) Autoclave homogenate to sterility at 121 degrees Celsius
for 1 hour (this autoclaving step can reduce viscosity for improved
layering).
[0145] 4) Cool to 22 degrees Celsius.
[0146] 5) Disperse 2.5 g particulate-ovalbumin in the 97.5 g, 22
degrees Celsius CMC-hydrogel and mix to homogeneity, under sterile
conditions.
[0147] 6) Vacuum treat the resulting material at 10 torr and 22
degrees Celsius for 2 hour to remove trapped micro-bubbles.
[0148] 7) Centrifuge product at 3,000 g for 1 hour in vacuum
chambered centrifuge (for floating and further removing residual
micro bubbles).
[0149] 8) Store the CMC-hydrogel product at 4 degrees Celsius.
[0150] Note in this example, particulate-ovalbumin is prepared from
activated iron beads reaction to ovalbumin. However, it should be
noted that the above descriptions are only exemplary embodiments
and other compounds and active ingredients can be used.
[0151] A solid block/sheet carboxymethylcellulose (CMC) can be
fabricated in the following manner using the low viscosity
CMC-hydrogels described above. The fabrication process can comprise
a laminar spreading of the polymer at a defined thickness and a
drying of the layered polymer to less than about 5% water content
using sterile dried air flow over the surface of the polymer layer.
The above two acts can repeated until the desired block thickness
is achieved.
[0152] A method of performing a laminar CMC-hydrogel layering of a
defined thickness over the casting mold assembly is described with
reference to FIG. 24. FIG. 24 illustrates a cross-sectional view of
the casting-mold assembly which includes: (a) casting bed; (b)
adjustable casting bed wall; (c) casting-bed depth adjustment
assembly; and (d) an acrylic spreader. It should be noted that FIG.
24 is not drawn to scale or otherwise shown with elements in their
proper proportions.
[0153] The casting mold assembly can be constructed from acrylic
(Plexiglas) and can comprise a casting bed base unit, a vertically
adjustable hydrophobic casting-bed wall, and a casting-bed
adjustment mechanism. The casting bed base unit (a1) can include a
removable/replaceable casting bed top plate (a2) with an attached
cellulose layer (a3). The cellulose layer can be about 0.5 mm in
thickness. The vertically adjustable hydrophobic casting-bed wall
(b) can be adjusted using the casting-bed depth adjustment
mechanism, which can be comprised of lead-screw (c1) and level
adjustment knob (c2). In the illustrated embodiment, a quarter turn
of this knob can result in a 0.5 mm lift of the bed wall.
[0154] Initially, the adjustable casting bed wall can be set to
height where the distance between the acrylic spreader and the
cellulose layer of the bed is about 1 mm when the spreader is in
position. A predefined volume (e.g., about 0.1 ml/cm2) of the 12.5%
CMC-hydrogel can be added and layered. The layer can be evened or
leveled by sliding the acrylic spreader (d) on the top surface of
the adjustable casting wall to yield an even layer of about 1 mm of
CMC-hydrogel. The layered CMC-hydrogel can be dried to a solid
phase in the drying apparatus shown in FIG. 25 and described in
more detail below.
[0155] The layering and drying steps can be repeated until the
desired layered structure (sheet) is achieved. The casting bed wall
can be raised by an appropriate amount during the addition of each
layer. For example, after adding each layer, the bed wall can be
raised or lifted by about 0.5 mm. Thus, the above-described cycle
can deposit about 0.5 mm solid CMC layer. The process (e.g., the
layering of material, the raising of bed wall, etc.) can be
repeated until the desired block thickness achieved. The layered
CMC-hydrogel polymer can be dried in various manners. For example,
FIG. 25 illustrates a drying apparatus that can be used to dry the
various deposited layers of the sheet material. It should be noted
that FIG. 25 is not drawn to scale or otherwise shown with elements
in their proper proportions. A fan can provide continuous gas flow
(e.g., air or other inert gas, such as nitrogen) over the
CMC-hydrogel layered in the casting mold assembly. The gas flow
will result in a gentle dehydration of the CMC-hydrogel layer. The
drying speed can be adjusted to prevent or reduce gas enclosures
(e.g., air bubbles) in the solid CMC product. The humid air over
the layer can be dried over desiccant (e.g., an air dryer or
dehumidifier), temperature adjusted, and returned over the hydrogel
again by the speed-controlled fan. A hygrometer can be positioned
on the humid side of the chamber to provide an indication of the
status of the drying process. After a predetermined dryness has
been achieved, as indicated by the hygrometer, the drying process
can be ended.
[0156] Airflow can be adjusted to affect the drying speed. In the
exemplary embodiment, the airflow is controlled to be between about
0.1-2.0 msec; the temperature is between ambient and about 50
degrees Celsius. Using these configurations, the drying time of a
single layer CMC-hydrogel can be about 0.5-4 hours depend on the
airflow and the set temperature.
[0157] The pure CMC based product can be transparent, light off
white, or amber colored. Its specific gravity can be about
1.55-1.58 g/ml. The product is desirably free of micro-bubbles and
otherwise suitable for fabricating micron scale objects. The
physical characterization of the final block/sheet product
(hardness, tensile strength, etc.) can vary, but should generally
be able to resist physical stresses associated with
micromilling.
[0158] As described above, the microneedle arrays disclosed herein
are capable of providing reliable and accurate delivery methods for
various bioactive components. The structural, manufacturing, and
distribution advantages characteristic of the above-described
microneedle arrays can be particularly applicable for use in
delivering vaccines. Advantages of these microneedle arrays include
(1) safety, obviating the use of needles or living vectors for
vaccine delivery, (2) economy, due to inexpensive production,
product stability, and ease of distribution, and 3) diversity, via
a delivery platform compatible with diverse antigen and adjuvant
formulations.
[0159] Moreover, cutaneous immunization by microneedle array has
important advantages in immunogenicity. The skin is rich in readily
accessible dendritic cells (DCs), and has long been regarded as a
highly immunogenic target for vaccine delivery. These dendritic
cell populations constitute the most powerful antigen presenting
cells (APCs) identified thus far. For example, genetic immunization
of skin results in transfection and activation of dendritic cells
in murine and human skin, and these transfected dendritic cells
synthesize transgenic antigens, migrate to skin draining lymph
nodes, and efficiently present them through the MHC class I
restricted pathway to stimulate CD8+ T-cells. The immune responses
induced by skin derived DCs are remarkably potent and long-lasting
compared to those induced by other immunization approaches. Recent
clinical studies demonstrate that even conventional vaccines are
significantly more potent when delivered intradermally, rather than
by standard intramuscular needle injection. Thus, microneedle
arrays can efficiently and simultaneously deliver both antigens and
adjuvants, enabling both the targeting of DCs and adjuvant
engineering of the immune response using the same delivery
platform.
[0160] In view of the many possible embodiments to which the
principles of the disclosed embodiments may be applied, it should
be recognized that the illustrated embodiments are only preferred
examples and should not be taken as limiting the scope of
protection. Rather, the scope of the protection is defined by the
following claims. We therefore claim all that comes within the
scope and spirit of these claims.
* * * * *