U.S. patent application number 15/936228 was filed with the patent office on 2018-09-27 for tip-loaded microneedle arrays for transdermal insertion.
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 | 20180272621 15/936228 |
Document ID | / |
Family ID | 49514855 |
Filed Date | 2018-09-27 |
United States Patent
Application |
20180272621 |
Kind Code |
A1 |
Falo, Jr.; Louis D. ; et
al. |
September 27, 2018 |
TIP-LOADED MICRONEEDLE ARRAYS FOR TRANSDERMAL INSERTION
Abstract
A method of forming a microneedle array can include forming a
microneedle array that has one or more bioactive component. The
microneedle array can include a base portion and plurality of
microneedles extending from the base portion, and the one or more
bioactive components are present in a higher concentration in the
plurality of microneedles than in 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
Pittsburgh
PA
Carnegie Mellon University
Pittsburgh
PA
|
Family ID: |
49514855 |
Appl. No.: |
15/936228 |
Filed: |
March 26, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14398375 |
Oct 31, 2014 |
9944019 |
|
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PCT/US2013/039084 |
May 1, 2013 |
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15936228 |
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61641209 |
May 1, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61M 37/0015 20130101;
A61L 31/042 20130101; B29C 67/00 20130101; A61M 2037/0053 20130101;
B23C 3/20 20130101; A61L 31/16 20130101; A61M 2037/0046 20130101;
A61K 9/0021 20130101; A61L 2400/18 20130101; A61B 2017/00526
20130101; B29L 2031/7544 20130101; A61L 2300/802 20130101 |
International
Class: |
B29C 67/00 20170101
B29C067/00; A61K 9/00 20060101 A61K009/00; A61L 31/04 20060101
A61L031/04; A61L 31/16 20060101 A61L031/16; A61M 37/00 20060101
A61M037/00; B23C 3/20 20060101 B23C003/20 |
Goverment Interests
ACKNOWLEDGMENT OF GOVERNMENT SUPPORT
[0002] This invention was made with government support under grant
numbers EB012776, AI076060, and CA121973 awarded by the National
Institutes of Health. The government has certain rights in the
invention.
Claims
1. A dissolvable microneedle array for transdermal insertion into a
patient comprising: one or more bioactive components; a base
portion; and a plurality of microneedles extending from the base
portion, wherein the one or more bioactive components are present
in a higher concentration in the plurality of microneedles than in
the base portion.
2. The microneedle array of claim 1, wherein substantially all of
the one or more bioactive components are located in the plurality
of microneedles so that the base portion is substantially formed
without any bioactive components contained therein.
3. The microneedle array of claim 2, wherein the one or more
bioactive components are locally concentrated in the plurality of
microneedles so that the one or more bioactive components are
generally present only in an upper half of respective microneedles
in the microneedle array.
4. The microneedle array of claim 2, wherein the plurality of
microneedles are pre-formed to have a shape that comprises 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, 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, wherein the
one or more bioactive components are substantially concentrated in
the area at or above the intermediate portion.
5. The microneedle array of claim 4, wherein each microneedle
generally tapers to a point above the intermediate portion and each
microneedle generally tapers to a smaller cross-sectional dimension
below the intermediate portion.
6. The microneedle array of claim 2, wherein each microneedle
comprises a plurality of layers of dissoluble biocompatible
material.
7. The microneedle array of claim 6, wherein the dissoluble
biocompatible material is carboxymethylcellulose.
8. The microneedle array of claim 2, wherein the one or more
bioactive component comprises at least two different bioactive
components.
9. The microneedle array of claim 8, wherein the at least two
different bioactive components are selected from the group
consisting of a chemotherapeutic agent, an adjuvant, and a chemo
attractant for a cancer chemo immunotherapy application.
10. The microneedle array of claim 9, wherein the bioactive
component comprises an antigen and an adjuvant for a vaccine
application.
11. The microneedle array of claim 2, wherein the one or more
bioactive component comprises at least one viral vector.
12. The microneedle array of claim 11, wherein the at least one
viral vector comprises an adenovector.
13. The microneedle array of claim 12, wherein the at least one
viral vector comprises an adenovector.
14. A method of fabricating a microneedle array comprising:
applying a first solution of a dissoluble biocompatible material
having one or more bioactive components contained therein to a
microneedle array production mold; applying a second solution of a
dissoluble biocompatible material that does not contain one or more
active components to the microneedle array production mold; and
drying the first and second solutions to form a solid microneedle
array that comprises a base portion and a plurality of microneedles
that extend from the base portion, wherein the one or more active
components are substantially concentrated in the plurality of
microneedles.
15. The method of claim 14, wherein the one or more bioactive
components are substantially concentrated in an upper half of
respective microneedles of the microneedle array.
16. The method of claim 14, wherein the one or more bioactive
components of the first solution comprises at least one viral
vector.
17. The method of claim 16, wherein the at least one viral vector
comprises an adenovector.
18. The method of claim 14, further comprising forming a sheet of
material having a plurality of layers formed by the application of
at least the first and second solutions.
19. The method of claim 18, wherein the method of forming the sheet
of material comprises spatially distributing the first solution
with the one or more bioactive component across the sheet of
material, and wherein the spatially distributed one or more
bioactive component is covered with the second solution of
dissoluble biocompatible material before removing portions from the
sheet of material to form the microneedle array.
20. The method of claim 18, wherein the at least one bioactive
component of the first solution or suspension comprises at least
one viral vector.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of U.S. patent
application Ser. No. 14/398,375, filed Oct. 31, 2014 and published
as U.S. Patent Publication No. 2015/0126923 which is the U.S.
National Stage of International Application No. PCT/US2013/039084,
filed May 1, 2013, which was published in English under PCT Article
21(2), which in turn claims the benefit of U.S. Provisional
Application No. 61/641,209, filed May 1, 2012. The previous
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. Current
fabrication approaches for dissolvable polymer-based microneedles
generally use microcasting processes. However, such conventional
processes are limited in the active components that can be embedded
into the array and are also wasteful in that they require that the
active components be homogenously embedded in the microneedles and
their support structures.
[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 and
fabrication 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] 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
[0011] FIG. 1 illustrates exemplary microneedles and their
dimensions.
[0012] FIG. 2 illustrates an exemplary microneedle array and its
dimensions.
[0013] FIGS. 3A and 3B illustrate exemplary microneedles with
tip-loaded active components.
[0014] FIGS. 4A and 4B illustrate exemplary microneedles with
tip-loaded active components.
[0015] FIGS. 5A and 5B illustrate exemplary microneedles with
tip-loaded active components.
[0016] FIGS. 6A and 6B illustrate exemplary microneedles with
tip-loaded active components.
[0017] FIG. 7 illustrates a miniature precision-micromilling system
used for fabricating microneedle mastermolds.
[0018] FIG. 8 is an SEM image of a micromilled mastermold with
pyramidal needles.
[0019] FIG. 9 is an SEM image of a pyramidal production mold.
[0020] FIG. 10 is an SEM image of an enlarged segment of the
production mold, illustrating a pyramidal needle molding well in
the center of the image.
[0021] FIGS. 11A-11D illustrate exemplary CMC-solids and embedded
active components.
[0022] FIGS. 12A-12B illustrate exemplary CMC-solids and embedded
active components.
[0023] FIG. 13 is a schematic illustration of exemplary vertical
multi-layered deposition structures and methods of fabricating the
same.
[0024] FIG. 14 is a schematic illustration of exemplary microneedle
arrays fabricated using layering and spatial distribution
techniques of embedded active components.
[0025] FIG. 15 is a schematic illustration of exemplary microneedle
arrays fabricated in a spatially controlled manner.
[0026] FIG. 16A is an SEM image of a plurality of pyramidal-type
molded microneedles.
[0027] FIG. 16B is an SEM image of a single pyramidal-type molded
microneedle.
[0028] FIG. 17 is an SEM image of a pillar type molded
microneedle.
[0029] FIG. 18 is a micrograph of pyramidal type molded
microneedles.
[0030] FIG. 19 is a micrograph of pillar type molded
microneedles.
[0031] FIG. 20 illustrates various microneedle geometries that can
be formed using micromilled mastermolds or by direct micromilling
of a block of material.
[0032] FIG. 21 illustrates a test apparatus for performing failure
and piercing tests.
[0033] FIGS. 22A and 22B illustrate force-displacement curves for
pillar type microneedles (22A) and pyramidal type microneedles
(22B).
[0034] FIG. 23 illustrates a finite elements model of microneedle
deflections for pillar type microneedles (left) and pyramidal type
microneedles (right).
[0035] FIGS. 24A-24F show various stereo micrographs of the
penetration of pyramidal (24A, 24C, 24E) and pillar (24B, 24D, 24F)
type microneedles in skin explants.
[0036] FIGS. 25A, 25B, and 25C illustrate the effectiveness of
microneedle arrays in penetrating skin explants.
[0037] FIGS. 26A and 26B illustrate in vivo delivery of
particulates to the skin draining lymph nodes of microneedle array
immunized mice.
[0038] FIG. 27 is a bar graph showing immunogenicity of microneedle
delivered model antigens.
[0039] FIG. 28 is a bar graph showing the stability of the active
cargo of CMC-microneedle arrays in storage.
[0040] FIGS. 29A and 29B show induction of apoptosis in epidermal
cells that have been delivered Cytoxan.RTM. (cyclophosphamide)
through a microneedle array.
[0041] FIG. 30 illustrates a microneedle geometry that can be
formed by direct micromilling of a block of material.
[0042] FIG. 31 is a stereo microscopic image of a direct-fabricated
solid CMC-microneedle array.
[0043] FIG. 32 is a stereo microscopic image of a portion of the
microneedle array of FIG. 31.
[0044] FIG. 33 is a schematic cross-sectional view of a
casting-mold assembly for creating a block or sheet of material for
direct micromilling.
[0045] FIG. 34 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.
[0046] FIG. 35 is a flow cytometry analysis of GFP expressing
target 293T cells.
[0047] FIG. 36 illustrates the stability of microneedle embedded
viruses after a number of days in storage.
[0048] FIG. 37 illustrates the expression and immunogenicity of
microneedle array delivered adenovectors.
[0049] FIG. 38 illustrates an applicator for microneedle insertion
into target tissue.
[0050] FIG. 39 illustrates applicator head designs for use with the
applicator shown in FIG. 38.
[0051] FIG. 40 is a schematic view of dimensional movement of an
applicator head.
DETAILED DESCRIPTION
[0052] 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.
[0053] 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 terms "biologic,"
"active component," "bioactive component," "bioactive material," or
"cargo" refer 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.
[0054] 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.
[0055] 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.
[0056] 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.
[0057] Tip-Loaded Microneedle Arrays
[0058] Dissolvable microneedle arrays enable efficient and safe
drug and vaccine delivery to the skin and mucosal surfaces.
However, inefficient drug delivery can result from the homogenous
nature of conventional microneedle array fabrication. Although the
drugs or other cargo that is to be delivered to the patient are
generally incorporated into the entire microneedle array matrix, in
practice only the microneedles enter the skin and therefore, only
cargo contained in the volume of the individual needles is
deliverable. Accordingly, the vast majority of the drugs or other
cargo that is localized in the non-needle components (e.g., the
supporting structure of the array) is never delivered to the
patient and is generally discarded as waste.
[0059] FIGS. 1 and 2 illustrate exemplary dimensions of
microneedles and microneedle arrays. Based on the illustrative
sizes shown in FIGS. 1 and 2, a microneedle array that comprises an
active component homogenously distributed throughout the array
exhibits active component waste of greater than 40 percent. For
example, if the entire area of the array is 61 mm.sup.2 and the
microneedle array area is 36 mm.sup.2, then the percent utilization
of the active component is less than 60 percent. Although the
dimensions reflected in FIGS. 1 and 2 illustrate a particular size
array and shape of microneedles, it should be understood that
similar waste is present in any other size microneedle array in
which the active component is homogenously distributed throughout
the array, regardless of the size of the array or the shape of the
microneedles involved.
[0060] The systems and methods described herein provide novel
microneedle array fabrication technology that utilizes a
fully-dissolvable microneedle array substrate and unique
microneedle geometries that enable effective delivery of a broad
range of active components, including a broad range of protein
and/or small molecule medicines and vaccines.
[0061] As described in more detail herein, in some embodiments,
this technology can also uniquely enable the simultaneous
co-delivery of multiple chemically distinct agents for
polyfunctional drug delivery. Examples of the utility of these
devices include, for example, (1) simultaneous delivery of multiple
antigens and adjuvants to generate a polyvalent immune response
relevant to infectious disease prevention and cancer therapy, (2)
co-delivery of chemotherapeutic agents, immune stimulators,
adjuvants, and antigens to enable simultaneous adjunct tumor
therapies, and (3) localized skin delivery of multiple therapeutic
agents without systemic exposure for the treatment of a wide
variety of skin diseases.
[0062] In some embodiments, the systems and method disclosed herein
relate to a novel fabrication technology that enables various
active components to be incorporated into the needle tips. Thus, by
localizing the active components in this manner, the remainder of
the microneedle array volume can be prepared using less expensive
matrix material that is non-active and generally regarded as safe.
The net result is greatly improved efficiency of drug delivery
based on (1) reduced waste of non-deliverable active components
incorporated into the non-needle portions of the microneedle array,
and (2) higher drug concentration in the skin penetrating needle
tips. This technological advance results in dramatically improved
economic feasibility proportional to the cost of drug cargo, and
increased effective cargo delivery capacity per needle of these
novel microneedle arrays.
[0063] FIGS. 3A, 3B, 4A, and 4B illustrate various embodiments of
microneedle arrays wherein the active component is concentrated in
the microneedle tips of the respective arrays. Thus, in contrast to
conventional microneedle arrays, the active component is not
present at even concentration throughout the microneedle array
since there is little or no active component present in the
supporting base structure. In addition, in some embodiments (as
shown, for example, in FIGS. 3A, 3B, 4A, and 4B), not only is there
little or no active component in the supporting structures, the
location of the active component is concentrated in the upper half
of the individual microneedles in the array.
[0064] FIGS. 5A and 5B illustrate exemplary images of microneedles
of a microneedle array that contains active component concentrated
in the upper half of the individual microneedles. The active
component is illustrated as fluorescent particles that are
concentrated in the tip of the microneedle, with the tip being
defined by an area of the microneedle that extends from a base
portion in a narrowing and/or tapered manner. The base portion, in
turn, extends from the supporting structure of the array.
[0065] FIGS. 6A and 6B illustrate additional exemplary images of
microneedles of microneedle arrays that contain active components
concentrated in the upper half of the individual microneedles. In
FIG. 6A, the active component, which is concentrated in the tip of
the microneedles, is BSA-FITC. In FIG. 6B, the active component,
which is also concentrated in the tip of the microneedles, is
OVA-FITC.
[0066] As noted above, in some embodiments, individual microneedles
can comprise active components only in the upper half of the
microneedle. In other embodiments, individual microneedles can
comprise active components only in the tips or in a narrowing
portion near the tip of the microneedle. In still other
embodiments, individual needles can comprise active components
throughout the entire microneedle portion that extends from the
supporting structure.
[0067] The following embodiments describe various exemplary methods
for fabricating microneedle arrays with one or more active
component concentrated in the upper halves and/or tips of
microneedles in respective microneedle arrays.
[0068] Microneedle Arrays Fabricated by Sequential Micro-Molding
and Spin-Drying Methods
[0069] The following steps describe an exemplary method of
fabricating microneedle arrays using sequential micro-molding and
spin-drying. Active components/cargo can be prepared at a desired
useful concentration in a compatible solvent. As described herein,
the solvents of the active component(s) can be cargo specific and
can comprise a broad range of liquids, including for example,
water, organic polar, and/or apolar liquids. Examples of active
components are discussed in more detail below and various
information about those active components, including tested and
maximum loading capacity of various microneedle arrays are also
discussed in more detail below.
[0070] If desired, multiple loading cycles can be performed to
achieve higher active cargo loads as necessary for specific
applications. In addition, multiple active cargos can be loaded in
a single loading cycle as a complex solution, or as single
solutions in multiple cycles (e.g., repeating the loading cycle
described below) as per specific cargo-compatibility requirements
of individual cargos. Also, particulate cargos (including those
with nano- and micro-sized geometries) can be prepared as
suspensions at the desired particle number/volume density.
Example 1
[0071] a) As described in more detail below in the micromilling
embodiments, an active cargo's working stock solution/suspension
can be applied to the surface of microneedle array production molds
at, for example, about 40 .mu.l per cm.sup.2 surface area.
[0072] b) The microneedle array production molds with active
cargo(s) can be centrifuged at 4500 rpm for 10 minutes to fill the
microneedle array production molds needles with the working cargo
stock.
[0073] c) The excess cargo solution/suspension can be removed and
the surface of the microneedle array production molds, washed with
100 .mu.l phosphate buffer saline (PBS) per cm.sup.2 mold-surface
area, or with the solvent used for the preparation of the active
cargo's working stock.
[0074] d) The microneedle array production molds containing the
active cargo stock solution/suspension in the needle's cavity can
be spin-dried at 3500 rpm for 30 minutes at the required
temperature with continues purging gas flow through the centrifuge
at 0-50 L/min to facilitate concentration of the drying active
cargo(s) in the needle-tips. The purging gas can be introduced into
the centrifuge chamber through tubular inlets. Moisture content can
be reduced using a dehumidifier tempered to the required
temperature with recirculation into the centrifuge chamber. The
purging gas can be air, nitrogen, carbon dioxide or another inert
or active gas as required for specific cargo(s). The flow rate is
measured by flow-meters and controlled by a circulating pump
device.
[0075] e) 100 .mu.l 20% CMC90 hydrogel in H2O can be added to the
surface microneedle array production molds' per cm.sup.2
microneedle array production molds-area to load the structural
component of the microneedle array device.
[0076] f) The microneedle array production molds can be centrifuged
at 4500 rpm for 10 min at the required temperature without purging
gas exchange in the centrifuge chamber to fill up the microneedle
array production molds needle cavities with the CMC90 hydrogel.
This can be followed by a 30 min incubation period to enable
rehydration of the active cargo(s) previously deposited in the
microneedle array tips.
[0077] g) The microneedle array production molds can centrifuged at
3500 rpm for 3 hours or longer at the required temperature with
0-50 L/min constant purging gas flow through the centrifuge chamber
to spin-dry the MNA devices to less than 5% moisture content.
[0078] h) The dried microneedle array devices can then be separated
from the microneedle array production molds for storage under the
desired conditions. In some embodiments, CMC90 based devices can be
storable between about 50.degree. C. to -86.degree. C.
[0079] Examples of fabricated tip-loaded active cargo carrying
microneedle arrays can be seen in FIGS. 3A-6B.
[0080] Micromilled Master Molds and Spin-Molded Microneedle
Arrays
[0081] In the following embodiments, micromilling steps are
preformed to create microneedle arrays of various specifications.
It should be understood, however, that the following embodiments
describe certain details of microneedle array fabrication that can
be applicable to processes of microneedle array fabrication that do
not involve micromilling steps, including the process described
above in the previous example.
[0082] In the following embodiments, 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. 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.
[0083] FIG. 7 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. As noted above, the micromilling process can be used to
create complex geometric features with many kinds of material.
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.
[0084] 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), 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.
[0085] 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.
[0086] 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.
[0087] 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.
[0088] FIG. 8 is an image from a scanning electron microscope (SEM)
showing the structure of a micromilled mastermold with a plurality
of pyramidal needles. As shown in FIG. 8, 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 of the production mold can
facilitate the spincasting processes discussed below. Although the
wall sections illustrated in FIG. 9 and the respective mastermold
structure shown in FIG. 8 is circular, it should be understood that
wall sections or containment means of other geometries can be
provided. For example, depending on what shape is desired for the
microneedle array device, the containment means can be formed in a
variety of shapes including, for example, square, rectangular,
trapezoidal, polygonal, or various irregular shapes.
[0089] As discussed above, the production molds can be made from
SYLGARD.RTM. 184 (Dow Corning), which is a two component clear
curable silicone elastomer that can be mixed at a 10:1 SYLGARD.RTM.
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. 9.). 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. or acrylic mastermolds.
[0090] FIG. 9 is an SEM image of a pyramidal production mold
created as described above. FIG. 10 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.
[0091] 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. As discussed above, each
microneedle can comprise bioactive components only in the
microneedles, or in some embodiments, only in the upper half of the
microneedles, or in other embodiments, only in a portion of the
microneedle that tapers near the tip. Thus, 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.
In the embodiments described herein, the portion without the
bioactive component includes the supporting structure of the
microneedle array and, in some embodiments, a base portion (e.g., a
lower half) of each microneedle in the array.
[0092] 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.
[0093] 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.
[0094] 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.
[0095] 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.
[0096] 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 Celsuis. The production molds can be
cleaned and reused for further casting of microneedle arrays.
[0097] In some embodiments, CMC-solids can be formed with layers
that do not contain active components and layers that contain
active components. FIGS. 11A-D illustrate CMC-solids with different
shapes (FIGS. 11A and 11B) and embedded active cargos on an upper
layer which becomes, after micromilling, the portions of the
microneedle with the active components. FIG. 11C illustrates micron
sized fluorescent particles layered on a surface of a non-active
component containing layer and FIG. 11D illustrates toluidine blue
examples layered on a surface of a non-active component containing
layer.
[0098] FIGS. 12A and 12B also illustrate CMC-solids with different
shapes, with FIG. 12B showing a square shape and FIG. 12B showing a
rectangular shape. Both CMC solids can be milled to dimensions for
further processing as described herein. It should be understood
that the geometries and the active cargo shown herein are not
intended to be limited to the exemplary embodiments.
Example 2
[0099] CMC-solids can be prepared with defined geometry and active
cargo contents in one or more layers of the prepared structure.
Examples of active cargos integrated into CMC-solids are described
more detail herein. Upon construction of the CMC-solids with
embedded active cargo contained in at least one layer of the
CMC-solid, the CMC solids can be milled to project-specific
dimensions and micro-milled to fabricate microneedle devices as
described herein.
Example 3
[0100] In another embodiment, one or more layers of active cargo
can be embedded on CMC-solids for direct micromilling of the
microneedle array. FIG. 13 illustrates a sample representation of
vertical multi-layered deposition and CMC embedding of active
cargos on CMC-solids for direct micro-milling of MNA devices.
[0101] In one exemplary method, microneedle arrays can be
fabricated by preparing CMC-solids with a defined geometries and
without any active cargo contained therein. Then, blank CMC-solids
can be milled to a desired dimension.
[0102] As shown in FIG. 13, active cargo(s) can be deposited onto
the CMC-solid in project specific geometric patterns for inclusion
of the active cargo(s) specifically in the tips of micro-milled MNA
devices.
[0103] The methods active cargo deposition onto the CMC-solid blank
can include, for example:
[0104] 1) Direct printing with micro-nozzle aided droplet
deposition.
[0105] 2) Transfer from preprinted matrices.
[0106] 3) Droplet-deposition with computer controlled robotic
systems.
[0107] FIG. 14 illustrates layering and spatial distribution of
embedded active cargos in a CMC-solid block. After the first layer
is deposited (A) it can be covered with a CMC layer (B) that
provides the surface for the subsequent deposition of the active
cargo (C). The process can be repeated until all desired layers are
deposited and encased in a solid CMC-block suitable for the
micro-milling process (D-F).
[0108] FIG. 15 illustrates a schematic view of a cross-section of a
CMC-block encasing the deposits of the active cargo in a spatially
controlled manner (A). The method allows 3-dimensional control and
placement of the active components after micro-milling in the
MNA-device (B). In panel (B) of FIG. 15, the placement of the
active cargos are shown in the stems of the active cargo; however
through the control of the milling process the placement can be
controlled vertically from the tip to the base of the microneedles.
Colors represent different active components or different
amount/concentration of the same material.
[0109] Thus, a method of vertically layered deposition of active
cargos in microneedles is provided by depositing one or more active
cargos sequentially on the surface of the CMC-solids in contact
with each other or separated by layers of CMC. In some embodiments,
horizontal pattern deposition of the active cargos can result in
spatial separation of the cargos. By combining vertical and
horizontal patterning of active cargo deposition, 3 dimensional
delivery and distribution of each of the defined active components
can be achieved, further reducing waste of active components during
fabrication of microneedle arrays.
[0110] Microneedle Integrated Adenovectors
[0111] The following embodiments are directed to dissolvable
microneedle arrays, such as those described herein, that
incorporate infectious viral vectors into the dissolvable matrix of
microneedle arrays. Using this technology, for the first time,
living viral vectors can be incorporated into microneedle arrays.
As described herein, the incorporation of viral vectors within the
disclosed microneedle arrays stabilizes the viral vectors so that
they maintain their infectivity after incorporation and after
prolonged periods of storage. The application of microneedle array
incorporated adenovectors (MIAs) to the skin results in
transfection of skin cells. In a vaccine setting, we have
demonstrated that skin application of MIAs encoding an HIV antigen
results in potent HIV specific immune responses. These results are
described in detail in the examples below.
Example 4
[0112] The microneedle integrated adenovectors preparation method
described herein preserves the viability of the adenoviral
particles during the preparation and in dry storage. These steps
were specifically designed based on the physical and chemical
properties of CMC microneedle arrays. Viral viability in CMC
microneedle arrays was achieved by [0113] Inclusion of low
viscosity carboxymethyl cellulose (CMC90) at 2.5% final
concentration (step 2.) and by [0114] Timed and temperature
controlled spin-drying concentration of the adenoviral particles in
the tips of the microneedle array devices (step 6.). [0115]
Controlled partial rehydration of the needle-tip loaded adenoviral
particles (step 8.)
[0116] Preparation of Tip-loaded Microneedle Integrated
Adenovectors (MIAs):
[0117] 1) Resuspend adenoviral particles at 2.times.109
particles/ml density in Trehalose-storage buffer (5% trehalose
Sigma-Aldrich USA, 20 mM Tris pH7.8, 75 mM NaCl, 2 mM MgCl2, 0.025%
Tween 80)
[0118] 2) Mix resuspended viral stock with equal volume of 5% CMC90
prepared in Trehasole-storage buffer, resulting in a 1.times.109
particles/ml density adenoviral working stock.
[0119] 3) Add adenoviral working stock suspension to the surface of
microneedle array production molds (as described in detail in other
embodiments herein) at 40 .mu.l per cm2 surface area.
[0120] 4) The molds are centrifuged at 4500 rpm for 10 minutes at
22.degree. C. to fill the needle tips with adenoviral working
stock.
[0121] 5) The excess viral stock is removed and the surface of the
molds washed with 100 .mu.l (phosphate buffer saline (PBS) solution
per cm2 mold-surface area.
[0122] 6) The microneedle array-molds containing the adenoviral
stock solution only in the needle's cavity are partially spin-dried
at 3500 rpm for 10 minutes at 22.degree. C.
[0123] 7) 100 .mu.l 20% structural, non-cargo containing CMC90
hydrogel in H2O added to the surface microneedle array-molds' per
cm2 mold-area to form the structure of the MIA device.
[0124] 8) Centrifuge at 4500 rpm for 10 min at 22.degree. C. to
fill up the needle cavities with 20% CMC90 and allow 30 min
incubation for the rehydration of the adenoviral particles dried in
the tips (step 3-6, above).
[0125] 9) By centrifugation spin-dry the MIA devices to less than
5% moisture content at 3500 rpm for 3 hours at 22.degree. C. with
10 L/min constant air flow through the centrifuge chamber.
[0126] 10) De-mold the dried MIA devices for storage at 4.degree.
C. or -80.degree. C.
Example 5
[0127] We have evaluated the potency and stability MNA incorporated
recombinant adenoviral particles. Ad5.EGFP was incorporated into
CMC hydrogel MNAs to fabricate a final product that contained 1010
virus particles/MNA. Control blank MNAs were prepared identically
but without the virus. Batches of Ad5.EGFP and control MNAs were
stored at RT, 4.degree. C. and at -86.degree. C. and viral
stability was evaluated in infectious assays. Specific transduction
activity of the MNA incorporated Ad5.EGFP virus was assessed in
vitro using 293T cells. Cells were plated at 2.times.106/well in
six well plates and transduced in duplicate with diluted virus
suspension, suspension+empty MNA (control), or Ad5.EGFP MNAs stored
at RT, 4.degree. C. and -86.degree. C. for the indicated time
periods. As a negative control untransduced wells were included.
Initially cell populations were analyzed after 24 h by flow
cytometry for GFP expression (representative histogram is shown in
FIG. 35.).
[0128] As shown in FIG. 35, the incorporation of Ad5.EGFP into MNAs
does not reduce transduction efficiency. Flow cytometry analysis of
GFP expressing target 293T cells 24 h after transduction with
identical titers of Ad5.EGFP either in suspension or incorporated
into CMC-patches vs. untransfected control cells. FIG. 36 shows the
stability of MNA embedded Ad5.EGFP virus. GFP gene expression was
assayed by flow cytometry as in FIG. 37 and normalized to the
infection efficiency of -86.degree. C. preserved Ad5.EGFP
suspension.
[0129] It has been found that the infection efficiency using MNA
Ad5.EGFP virus was 87.92.+-.4.5%, which is similar to that observed
for traditional -86.degree. C. preserved Ad5.EGFP suspension (FIGS.
35 and 36), suggesting that the manufacturing process does not
adversely affect the transduction efficiency of Ad-EGFP viral
particles. To assess infectivity over time, the transfection
efficiency of freshly prepared -86.degree. C. preserved Ad5.EGFP
suspensions was compared to that of MNA incorporated Ad5.EGFP
stored for prolonged periods of time at either RT, 4 C, or -86 C.
Infectivity (normalized to Ad5.EGFP suspension+empty CMC-patch) is
reported for storage periods of up to 365 days (FIG. 36). These
results suggest that the infectiousness of MNA Ad5.EGFP is
remarkably stable with storage at either 4 C or -86 C, and somewhat
stable at RT for up to 30 days.
[0130] These results demonstrate that microneedle array delivered
Ad transgenes are expressed in the skin and induce potent cellular
immune responses. To specifically evaluate gene expression in vivo,
we determined GFP expression in skin following either traditional
intradermal injection (I.D.) or microneedle array-mediated
intracutaneous delivery. We delivered 108 Ad5.GFP viral particles
by ID injection or topically via a single microneedle array
application (FIG. 37). Skin was harvested 48 h later,
cryosectioned, counter-stained using blue fluorescent DAPI to
identify cell nuclei, and then imaged by fluorescent microscopy.
Significant cellular GFP expression was observed following both
I.D. and microneedle array delivery. To evaluate immunogenicity, we
evaluated antigen-specific lytic activity in vivo following a
single I.D. or microneedle array immunization without boosting. For
this purpose we immunized groups of mice with E1/E3-deleted
Ad5-based vectors that encode codon-optimized SIVmac239 gag
full-length or SIVmac239 gag p17 antigens (Ad5.SIV gag, Ad5.SIV gag
p17). Empty vector was used as a control (Ad5). We observed potent
and similar levels of in vivo lytic activity specific for the
dominant SIVgag p17-derived peptide KSLYNTVCV (SIVmac239 gag 76-84)
following either I.D. or microneedle array immunization with either
Ad5.SIV gag or Ad5.SIV gag p17 (FIG. 37, CTL).
[0131] The microneedle array technology disclosed herein can also
facilitate clinical gene therapy. It addresses, for example, at
least two major limitations of conventional approaches. First, it
enables stabilization and storage of recombinant viral vectors for
prolonged periods of time. By rendering live virus vectors
resistant to high and low temperatures with proven seroequivalence
to frozen liquid formulations, microneedle array stabilization will
relieve pressures related to the `cold chain.` Further, integration
in microneedle arrays enables precise, consistent and reproducible
dosing of viral vectors not achievable by conventional methods.
Finally, the viral vector is repackaged in the only necessary
delivery device, the biocompatible and completely disposable
microneedle array that directs delivery precisely to the
superficial layers of the skin.
[0132] Such a gene delivery platform is useful in providing
patient-friendly, clinical gene therapy. Since these microneedle
arrays have been engineered to not penetrate to the depth of
vascular or neural structures, gene delivery to human skin will be
both painless and bloodless. In addition, the fabrication process
is flexible, enabling simple and rapid low cost production with
efficient scale-up potential. Also, as a final product, the MIA
device it is stable at room temperature and is inexpensive to
transport and store. In combination, these structural and
manufacturing advantages can enable broad and rapid clinical
deployment, making this gene delivery technology readily applicable
to the prevention and/or treatment of a broad range of human
diseases. Moreover, this approach can be extended to other
vector-based vaccine platforms that are currently restricted by the
same limitations (e.g., vaccinia virus, AAV etc.). For at least
these reasons, the disclosed microneedle arrays and methods of
using the same significantly advance the recombinant gene therapy
field.
[0133] Microneedle Arrays--Exemplary Active Components
[0134] Various active components are described in detail below. For
convenience, the following examples are based on an microneedle
array which is 6.3.times.6.3 mm. This size, and hence cargo
delivery can be varied by increasing or decreasing 2-100 fold.
[0135] General considerations for the maximum active cargo
quantities include, for example, total needle volume in the array
and solubility of the active component(s) in the solvent (generally
expected to be <50%).
TABLE-US-00001 Tip Loaded Amount Tip Loaded Max. predicted
Components: into MNA device loading capacity .mu.g/device (unless
indicated differently)
TABLE-US-00002 Live viruses.sup.(1) Ad5.GFP 5 .times. 10.sup.8 2-5
.times. 10.sup.9 (adeno viral particles/MNA particles/MNA GFP
expression vector) Ad-SIVgag 5 .times. 10.sup.8 2-5 .times.
10.sup.9 (adeno viral particles/MNA particles/MNA gag expression
vector) Ad-SIVp17 5 .times. 10.sup.8 2-5 .times. 10.sup.9 (adeno
viral particles/MNA particles/MNA gag-p17 expression vector) .PSI.5
5 .times. 10.sup.8 2-5 .times. 10.sup.9 (non-recombinant Ad
particles/MNA particles/MNA vector) Lenti-GFP.sup.(2) 5 .times.
10.sup.6 2-5 .times. 10.sup.7 (Lenti viral GFP particles/MNA
particles/MNA expression vector)
Vaccinia virus (immunization) Recombinant vaccinia virus (gene
therapy, genetic engineering) Seasonal influenza
MMR (Measles, Mumps, Rubella)
TABLE-US-00003 [0136] Proteins/Peptides BSA (FITC labeled) 240 400
OVA (FITC labeled) 100 400 OVA (no label) 240 400 Flu (split
vaccine) 0.22 (2-5)
TABLE-US-00004 Epitope Peptides.sup.(3) TRP-2 50 200 EphA2 (a) 50
400 EphA2 (b) 50 400 DLK-1 50 200 Multiple epitopes 200 400-600 in
one MNA Substance-P 15 (NK-1R ligand)
TABLE-US-00005 Nucleic acids CpG 1668 120 250 CpG 2006 120 250
Poly(I:C) 250 250 Plasmid vectors 100 200 (High mol. weight
DNA)
TABLE-US-00006 Peptides/Nucleic acid combos OVA/CpG 250/120
OVA/CpG/poly(I:C) 250/120/250 Epitope 200/250
peptides/poly(I:C)
TABLE-US-00007 Organics Doxorubicin 100 R848 (TLR7/8 ligand) 6 L733
2 (NK-1 antagonist) DNCB (irritant) 100
TABLE-US-00008 Particulates Micro-particles 1 .times. 10.sup.6 2-5
.times. 10.sup.7 (1 .mu. diameter particles/MNA particles/MNA
microsphares)
Nano scale particles PLG/PLA based
TABLE-US-00009 Other Biologic tumor lysate/CpG 250/120 tumor
lysate/CpG/poly(I:C) 250/120/250 tumor lysates/poly(I:C)
200/250
Tip-loading of live adenoviruses generally includes the following
modifications: [0137] a) The presence of 5% trehalose and 2.5%
CMC90 in the tip-loading hydrogel suspension. [0138] b) The
temperature of the process is maintained at 22.degree. C. In
addition, Lenti viral vectors generally require 4.degree. C.
processing and vapor trap based humidity controls. Also, short
epitope peptides generally are solubilized in DMSO, with the
evaporation time of the solvent during tip-loading is 4 hours.
[0139] Microneedle Structures and Shapes
[0140] For each of the embodiments below, it should be understood
that one or more layers of active components can be provided in the
microneedles of the microneedle arrays as described above. Thus,
for example, in some embodiments, active components are only
provided in the area of the microneedle--not in the structural
support of the array, such as shown in FIG. 15. Moreover, in other
embodiments, the active components are concentrated in the upper
half of the microneedles, such as in the tips of the microneedles
as shown in FIGS. 3A-4B.
[0141] FIGS. 16A and 16B 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. 16A is about 5-10 .mu.m. As shown in FIG. 16B, the
sides of the pyramidal needles can be formed with curved and/or
arcuate faces that can facilitate insertion in skin.
[0142] FIG. 17 is another SEM image of a single needle of a
microneedle array. The microneedle shown in FIG. 17 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.
[0143] FIGS. 18 and 19 illustrate micrographs of pyramidal (FIG.
18) and pillar type (FIG. 19) 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.
[0144] 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.
[0145] 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.
[0146] 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.
[0147] 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.
[0148] Mechanical Integrity and Penetration Capabilities
[0149] 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.
[0150] FIG. 21 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.
[0151] FIGS. 22A and 22B illustrate force-displacement curves of
data measured during failure tests. FIG. 22A is representative of
data obtained from testing a pillar microneedle sample and FIG. 22B
is representative of data obtained from testing a pyramid
microneedle. As seen in FIGS. 22A and 22B, 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).
[0152] 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.
[0153] To further analyze the failure of the microneedles, the
finite-elements model (FEM) of the microneedle arrays shown in FIG.
23 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.
[0154] 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.
[0155] 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.
[0156] 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. FIGS. 24A-24F illustrate 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.
[0157] 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.
[0158] 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 (FIGS. 24C vs. 24D).
Images of the recovered patches (FIGS. 24A vs. 24B) 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
(FIGS. 24E vs. 24F).
[0159] To further evaluate penetration and to assess delivery
effectiveness to human skin, CMC-microneedle arrays were fabricated
with BioMag (Polysciences, Inc., cat#. 84100) beads or fluorescent
particulate tracers (Fluoresbrite 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. 25A and 25B) or by fluorescent
microscopy (FIG. 25C).
[0160] 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. 25A and 25B). In
particular, ordered cavities (FIG. 25A, cavities numbered 1-4,
toluene blue counterstain, 10.times.) and deposits of BioMag
particles (brown) lining penetration cavities were evident (FIG.
25B, 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. 25C, DAPI (blue), HLA-DR+ (red) and
fluorescent particles (green), 40.times.).
[0161] 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.
[0162] 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. 26A and 26B illustrates
substantial numbers of particles that were evident in the draining
lymph Nodes (FIG. 26A, 10.times.), including clusters of
particulates closely associated with Class II+ cells (FIG. 26B,
60.times.) suggesting the presence of lymph node resident antigen
presenting cells with internalized particulates.
[0163] 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.106 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.
[0164] 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-00010 TABLE 4.2.5 Transfer of .sup.3H-labeled
CMC-microneedle material into human skin explants by pyramidal and
pillar-type needles. Pyramid Pyramidal Needles Pillar-Type
Pillar-Type Needles Pillar to Array Needles STDev OVA Transferred
Needles STDev OVA 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 3068.06 19.00 2.12 11919.63 6.77 8.24 3.89 AVG
[0165] 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.
[0166] 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.
[0167] 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.
[0168] 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.
[0169] In an exemplary embodiment, the delivery of a biologically
active small molecule was studied. In particular, the activity of
the chemotherapeutic agent Cytoxan.RTM. delivered to the skin with
CMC microneedle arrays was studied. The use of Cytoxan.RTM. enables
direct measurement of biologic activity (Cytoxan.RTM. 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.
[0170] 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.
[0171] 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.
[0172] 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. 27). 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.
[0173] 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. 28, 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.
[0174] 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 FluoresBrite green fluorescent
particles as a control. Cytoxan.RTM. 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..
Cytoxan.RTM. 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. treated skin as shown in FIG. 29A. As shown in FIG.
29B, 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.
[0175] Direct Fabricated Microneedle Arrays
[0176] 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.
[0177] 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.
[0178] 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.
[0179] 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.
[0180] 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. 30
illustrates a beveled, undercut microneedle shape that cannot be
molded in the manners described herein.
[0181] 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. 30 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.
[0182] 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.
[0183] 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.
[0184] FIG. 31 is a stereo-microscopic image analysis of an entire
microneedle array. The microneedle comprises a 10.times.10 array of
microneedles. FIG. 32 is an enlarged segment of the microneedle
array of FIG. 31. The layering of two components is shown in FIG.
32, 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.
[0185] 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. 30), which can
significantly improve the retained needle-volume and needle
retention time in the skin.
[0186] 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.
[0187] 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.
[0188] 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.
[0189] The preparation of 1000 g sterile 12.5% CMC hydrogel with no
active component can be achieved as follows:
[0190] 1) Measure 125 g CMC, add 875 g water or other water based
solvent.
[0191] 2) Stir to homogeneity in overhead mixer.
[0192] 3) Autoclave homogenate to sterility at 121 degrees Celsius
for 1 hour (the autoclaving step can reduce viscosity for improved
layering)
[0193] 4) Cool to 22 degrees Celsius.
[0194] 5) Vacuum treat the resulting material at 10 torr and 22
degrees Celsius for 1 hour to remove trapped micro-bubbles.
[0195] 6) Centrifuge product at 25,000 g for 1 hour in vacuum
chambered centrifuge (for floating and further removing residual
micro bubbles).
[0196] 7) Store the CMC-hydrogel product at 4 degrees Celsius.
[0197] The preparation of 1000 g sterile 12.5 w/w % dry content
20/80% ovalbumin/CMC hydrogel can be achieved as follows:
[0198] 1) Measure 100 g CMC add 650 g water or other water based
solvent.
[0199] 2) Stir to homogeneity in overhead mixer.
[0200] 3) Autoclave homogenate to sterility at 121 degrees Celsius
for 1 hour (this autoclaving step can reduce viscosity for improved
layering).
[0201] 4) Cool to 22 degrees Celsius.
[0202] 5a) Dissolve 25 g ovalbumin in 225 g water.
[0203] 5b) Sterile filter ovalbumin solution on 0.22 .mu.m pore
sized filter.
[0204] 6) Mix to homogeneity, under sterile conditions the 750 g
CMC hydrogel with 250 g sterile ovalbumin solution.
[0205] 7) Vacuum treat the resulting material at 10 torr and 22
degrees Celsius for 1 hour to remove trapped micro-bubbles.
[0206] 8) Centrifuge product at 25,000 g for 1 hour in vacuum
chambered centrifuge (for floating and further removing residual
micro bubbles).
[0207] 9) Store the CMC-hydrogel product at 4 degrees Celsius.
[0208] The preparation of 100 g sterile 12.5 w/w % dry content
20/80% particulate-ovalbumin/CMC hydrogel can be achieved as
follows:
[0209] 1) Measure 10 g CMC add 87.5 g water or other water based
solvent.
[0210] 2) Stir to homogeneity in overhead mixer.
[0211] 3) Autoclave homogenate to sterility at 121 degrees Celsius
for 1 hour (this autoclaving step can reduce viscosity for improved
layering).
[0212] 4) Cool to 22 degrees Celsius.
[0213] 5) Disperse 2.5 g particulate-ovalbumin in the 97.5 g, 22
degrees Celsius CMC-hydrogel and mix to homogeneity, under sterile
conditions.
[0214] 6) Vacuum treat the resulting material at 10 torr and 22
degrees Celsius for 2 hour to remove trapped micro-bubbles.
[0215] 7) Centrifuge product at 3,000 g for 1 hour in vacuum
chambered centrifuge (for floating and further removing residual
micro bubbles).
[0216] 8) Store the CMC-hydrogel product at 4 degrees Celsius.
[0217] 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.
[0218] 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.
[0219] A method of performing a laminar CMC-hydrogel layering of a
defined thickness over the casting mold assembly is described with
reference to FIG. 33. FIG. 33 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.
33 is not drawn to scale or otherwise shown with elements in their
proper proportions.
[0220] 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.
[0221] 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. 34 and described in
more detail below.
[0222] 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.
[0223] The layered CMC-hydrogel polymer can be dried in various
manners. For example, FIG. 34 illustrates a drying apparatus that
can be used to dry the various deposited layers of the sheet
material. It should be noted that FIG. 34 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.
[0224] 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 m/sec; 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.
[0225] 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.
[0226] 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.
[0227] 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.
[0228] High Frequency Electro-magnetic Oscillating Applicator
[0229] Microneedle array devices can be applied to human skin by a
variety of methods including self or assisted application by human
pressure (e.g., pushing with a finger or thumb), or with
spring-loaded devices. To facilitate the ease and reproducibility
of delivery of microneedle array devices, including tip-loaded
microneedle arrays, an applicator device is described herein. The
applicator device is configured to convert high frequency
electromagnetic oscillation into unidirectional mechanical
resonance of the active head. This in turn enables multiple
reproducible low amplitude and high frequency pressure strokes that
facilitate insertion of the microneedles of microneedle arrays into
tissues including human skin.
[0230] As shown in FIG. 38, the applicator can comprise an
applicator head, an oscillator-energy converter, an
electro-magnetic oscillator, and a power source. If desired, one,
or all of these four elements can be detachable from the applicator
device.
[0231] An applicator head can be interchangeable to accommodate and
act on different sized and shaped tissue surface areas. As shown in
FIG. 39, various applicator head geometries can be utilized in
combination with the applicator described herein. Applicator heads
are interchangeable made of stainless steel or other chemically and
physically resistant materials. If desired, the applicator heads
can be autoclaved for sterility and/or sterilized by alcohols or
other chemicals. Alternatively, or in addition, gas sterilization
(ethylene oxide) is possible.
[0232] Application specific geometries can be rapidly designed and
fabricated. For example, the area of a single application in this
example can range from 5 mm.sup.2 to 250 mm.sup.2 dependent on the
active head's geometry. A broader range can be achieved by simple
structural variation of the head's geometry.
[0233] The oscillator energy converter unit can be configured to
transforms the electro-magnetic oscillation into mechanical
movements of the applicator head. The amplitude of the applicator
head's in direction Z can be controlled between 0.5-2.5 mm (FIG.
40; A). In some embodiments, head movements in direction X-Y can be
configured to be negligible, <0.2 mm (FIG. 40; B). The frequency
of the mechanical movements resulting from the energy conversion in
the direction Z can be controlled between 500-25000 rpm. If
desired, the oscillator energy converter unit can be detachable and
can be disposed or sterilized as needed.
[0234] The electro-magnetic (EM) oscillator can be composed of
three subunits. These subunits can include a (1) regulated power
supply generating the voltage and power for the controller and high
frequency EM oscillator; a (2) controller-regulator generates the
high frequency signal and the required current for the EM
oscillator; and (3) an EM oscillator. The output frequency can be
controlled by the user (e.g., in ranges such as from 100-500 Hz).
In some embodiments, the EM oscillator can be fully enclosed and
can be sterilized by alcohol solutions or other chemical
agents.
[0235] The power source unit can also be detachable to accommodate
different attachable power sources such as:
[0236] a. Battery, regular disposable alkaline or any other
type.
[0237] b. Rechargeable NiCad or Li oxide battery with built in
inductive charger.
[0238] c. Electronic power adapter to 100-240 V
[0239] The applicator can provide several benefits in connection
with microneedle array application. For example, the applicator can
minimalize the mechanical force needed for microneedle array
insertion into tissues. The applicator can also reduce pain effects
compared to existing spring-loaded applicators. In addition, the
applicator can be portable and the components of the applicator can
be detachable and interchangeable. Finally, the applicator can be
configured so that it is capable of being sterilized for aseptic
use.
[0240] 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.
* * * * *