U.S. patent application number 17/326064 was filed with the patent office on 2022-02-10 for method of delivering material or stimulus to a biological subject.
The applicant listed for this patent is Vaxxas Pty Limited. Invention is credited to Mark Anthony Fernance KENDALL.
Application Number | 20220039812 17/326064 |
Document ID | / |
Family ID | 1000005925921 |
Filed Date | 2022-02-10 |
United States Patent
Application |
20220039812 |
Kind Code |
A1 |
KENDALL; Mark Anthony
Fernance |
February 10, 2022 |
METHOD OF DELIVERING MATERIAL OR STIMULUS TO A BIOLOGICAL
SUBJECT
Abstract
A device for delivery of material or stimulus to targets within
a body to produce a desired response, the targets being at least
one of cells of interest, cell organelles of interest and cell
nuclei of interest. The device includes a number of projections for
penetrating a body surface, with the number of projections being
selected to produce a desired response, and the number being at
least 500. A spacing between projections is also at least partially
determined based on an arrangement of the targets within the
body.
Inventors: |
KENDALL; Mark Anthony Fernance;
(Queensland, AU) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Vaxxas Pty Limited |
Sydney |
|
AU |
|
|
Family ID: |
1000005925921 |
Appl. No.: |
17/326064 |
Filed: |
May 20, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15849134 |
Dec 20, 2017 |
11207086 |
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17326064 |
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13251920 |
Oct 3, 2011 |
9888932 |
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15849134 |
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11496053 |
Jul 27, 2006 |
8052633 |
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13251920 |
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PCT/GB2005/000336 |
Jan 31, 2005 |
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11496053 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61M 37/0015 20130101;
A61B 17/205 20130101; A61M 2037/0046 20130101; B33Y 80/00
20141201 |
International
Class: |
A61B 17/20 20060101
A61B017/20; A61M 37/00 20060101 A61M037/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 30, 2004 |
GB |
0402131.7 |
Claims
1.-45. (canceled)
46. A method of designing a microprojection array targeting a
particular target in a subject's skin comprising microprojections
having a targeting section and a support section comprising:
selecting a target below the surface of the skin in a layer of the
skin wherein the skin layer has an upper portion and a lower
portion; selecting a diameter of the targeting section to be
smaller than the diameter of the target; selecting a
microprojection length based on the target depth below the skin
such that the microprojection penetrates the upper portion of the
layer but does not exceed the lower portion of the layer;
determining the likelihood of a single targeting event; determining
the number of targeting events required for a desired effect;
determining the number of microprojections; determining the spacing
between microprojections based on the target diameter and target
spacing; and combining the information to design a microprojection
array targeting a particular target in a subject's skin.
47. The method of claim 46 wherein the target is Langerhans
cells.
48. The method of claim 47 wherein the desired effect is an
immunological response in the subject
49. The method of claim 48 wherein the length of the support
section is:
(D.sub.c+h.sub.layer/2).gtoreq.a.gtoreq.(D.sub.c-h.sub.layer/2)
where: D.sub.c is the depth of the target layer; h.sub.layer is the
height of the layer in the skin; and, a is the length of the
support section.
50. The method of claim 49 wherein the length of the targeting
section is l.gtoreq.h.sub.layer.
51. The method of claim 49 wherein the likelihood of a single
target event is determined by: P depth = .intg. a - Q.sigma. l + a
- Q.sigma. .times. dx .sigma. .times. 2 .times. .pi. .times. e - (
x - D c .sigma. ) - 2 ##EQU00012## where: .sigma. is the standard
deviation from a mean location, accounting for the skin surface
undulations. D.sub.c is a distance of the cells of interest below a
surface of the body against which the device is to be applied in
use; Q is a number of standard deviations from a mean level of the
surface of the body at which the device comes to rest in use; a is
the length of the support section; and, l is the length of the
targeting section.
52. The method of claim 51 wherein the spacing between the
microprojections is less than or equal to the diameter of the
target.
Description
CROSS-REFERENCE TO RELATED APPLICATION(S)
[0001] The current application is a continuation of U.S. Ser. No.
11/496,053 filed on Jul. 27, 2006, which is a continuation-in-part
(CIP) of International Application Number PCT/GB2005/000336 which
designates the U.S. and has an International Filing Date of 31 Jan.
2005 and which was published as International Publication No.
WO2005/072630 on 11 Aug. 2005. The entirety of International
Application Number PCT/GB2005/000336 is hereby incorporated herein
by reference.
BACKGROUND
Technical Field
[0002] The present invention relates to a device for delivery of
material or stimulus to targets within a body to produce a desired
response, and in particular to a device including a number of
projections for penetrating a body surface. The invention can also
relate to devices for delivering bioactive substances and other
stimuli to living cells, to methods of manufacture of the device
and to various uses of the device.
Description of the Related Art
[0003] The reference in this specification to any prior publication
(or information derived from it), or to any matter which is known,
is not, and should not be taken as an acknowledgment or admission
or any form of suggestion that the prior publication (or
information derived from it) or known matter forms part of the
common general knowledge in the field of endeavor to which this
specification relates.
[0004] In recent years, attempts have been made to devise new
methods of delivering drugs and other bioactive materials, for
vaccination and other purposes, which provide alternatives that are
more convenient and/or enhanced in performance to the customary
routes of administration such as intramuscular and intradermal
injection. Limitations of intradermal injection include:
cross-contamination through needle-stick injuries in health
workers; injection phobia from a needle and syringe; and most
importantly, as a result of its comparatively large scale and
method of administration, the needle and syringe cannot target key
cells in the outer skin layers (FIG. 11(a)). This is a serious
limitation to many existing and emerging strategies for the
prevention, treatment and monitoring of a range of untreatable
diseases.
[0005] The skin structure is shown in FIG. 11, with a summary of
key existing delivery methods. Non-invasive methods of delivery
through the skin have been used, including patches, liquid
solutions and creams. Their success is dependent upon the ability
to breach the semi-permeable stratum corneum (SC) into the viable
epidermis. Typically, larger biomolecules are unable to breach this
barrier.
[0006] Alternatively, there are many more "invasive" means to
breach the SC for pharmaceutical delivery to the viable epidermis.
Simple methods include: tape stripping with an abrasive tape to or
sandpaper and the application of depilatory agents. Amongst the
more advanced technologies are electroporation, ablation by laser
or heat, radiofrequency high voltage currents, iontopheresis,
liposomes, sonophoresis. Many of these approaches remain untested
for complex entities such as vaccines and immunotherapies.
Moreover, they do not specifically deliver entities within key skin
cells.
[0007] Needle-free injection approaches include the high-speed
liquid jet injector, which had a rise and fall in popularity in the
mid twentieth century-and has recently seen a resurgence (Furth, P.
A., Shamay, A. & Henninghausen, L. (1995) Gene transfer into
mammalian cells by jet injection. Hybridoma, 14:149-152.). However,
this method delivers jets of liquid to the epidermis and dermis
(labelled (c) in Fig A), usually with a diameter >100 .mu.m and
not within key cells. Furthermore, as a result of the concentrated
jet momentum, many skin cells die. Delivery into the dermis also
leads to patients reporting pain from injection.
[0008] The ballistic, needle-free delivery of microparticles (or
gene gun) offers a route for delivering biological agents directly
into cells of the skin. In this needle-free technique,
pharmaceutical or immunomodulatory agents, formulated as or coated
to particles, are accelerated in a high-speed gas jet at sufficient
momentum to penetrate the skin (or mucosal) layer and to achieve a
pharmacological effect. A schematic of microparticles in the skin
following ballistic delivery is shown in FIG. 11(b). The ability of
this "scatter gun" approach to deliver genes and drugs to epidermal
cells is highly limited and sensitive to biological variability in
skin properties on the dynamic high strain rate ballistics process.
These effects are discussed in Kendall, M. A. F., Rishworth, S.,
Carter, F. V. & Mitchell, T. J. (2004) "The effects of relative
humidity and ambient temperature on the ballistic delivery of
micro-particles into excised porcine skin." J. Investigative
Dermatology, 122(3):739-746.).; and Kendall, M. A. F., Mitchell, T.
J. & Wrighton-Smith, P. (2004) Intradermal ballistic delivery
of microparticles into excised human skin for drug and vaccine
applications. J. Biomechanics, 37(11):1733-1741.
[0009] First, the ballistic delivery of particles into the skin to
target epidermal cells is extremely sensitive to the small
variations in the stratum corneum-including the stratum corneum
thickness, which varies massively with body site, age, sex, race
and exposure to climatic conditions. (The quasi-static loading of
skin with micro-nanostructures would be less sensitive to these
differences).
[0010] Second, it has been shown that even when all these
parameters are strictly controlled-and the only parameter varied is
the climatic relative humidity (15%-95%), or, independently,
temperature (20.degree. C.-40.degree. C.)-the result is a large
variation in penetration depth. These results are shown in FIG. 12,
with particle penetration as a function of ambient relative
humidity (FIG. 12A) and ambient temperature (FIG. 12B) plotted
along with theoretical calculations of particle penetration and
measured stratum corneum thickness. This variation alone is
significant and sufficient to make the difference between particles
breaching the stratum corneum, or not.
[0011] The compound effect of these two (and other) sources of
variability is the gene gun/biolistics process does not
consistently target epidermal cells-leading to inconsistent
biological responses (e.g., in DNA vaccination).
[0012] Interestingly, it has also been shown that the high
strain-rate loading of the skin under ballistic particle impact
(approximately 10.sup.6 per second) increases the stratum corneum
breaking stress by up to a factor of 10 compared to quasi-static
values-due to a ductile-to-brittle change in the skin mechanical
properties. This means that the tissue is more difficult to
penetrate as the particle impact velocity is increased. Therefore
it is desirable to devise a way to deliver micro/nanostructures to
the skin at lower strain-rates than the ballistic approach to
exploit the weaker stratum corneum.
[0013] When the microparticles are delivered to the skin, it is
unclear whether there are any adverse longer term effects. For
example, in the case of insoluble particles, many of them slough
off with the usual skin turnover. However, gold particles have been
detected in the lymph nodes following ballistic particle
delivery-presumably by migration with Langerhans cells. Uncertainty
of adverse effects of these delivered materials would be removed by
delivery routes that do not leave such materials in tissue
site.
[0014] Moreover, when the microparticles successfully target cells,
there is a significant probability they kill the cells they target.
Consider a typical ballistic delivery condition: over 1 million 2-3
um diameter gold particles coated in DNA to the skin at 400-600
m/s, over a target diameter of 4 mm (Kendall, M. A. F., Mulholland,
W. J., Tirlapur, U. K., Arbuthnott, E. S. & Armitage, M. (2003)
Targeted delivery of micro-particles to epithelial cells for
immunotherapy and vaccines: an experimental and probabilistic
study. 6th International Conference on Cellular Engineering. Aug.
20-22 2003, Sydney, Australia.). Reported experiments with these
conditions using cell death stains (ethidium bromide/acridin
orange) show that microparticles impacting the skin do kill cells
(McSloy, N. J., Raju, P. A. & Kendall, M. A. F. (2004) The
effects of shock waves and particle penetration in skin on cell
viability following gene gun delivery. British Society for Gene
Therapy, 1st Annual Conference. Oxford, UK, Mar. 28-30 2004; Raju,
P. A. & Kendall, M. A. F. (2004) Epidermal cell viability
following the ballistic delivery of DNA vaccine microparticles. DNA
Vaccines2004-the Gene Vaccine Conference. 17-19 Nov. 2004, Monte
Carlo, Monaco.).
[0015] FIG. 13A shows the percentage of cells that had membrane
rupture (i.e., death) as a function of the localized particle
channel density. In FIG. 13B we see schematically the way the data
in FIG. 13A was achieved, relating "tracks" left by particle
penetration to the death of cells in a layer of the viable
epidermis. Clearly, FIG. 13A shows that at a channel density above
0.01 channels/micron, all the cells in that layer are dead. Indeed,
FIG. 13C shows that cells are killed when the particle is passing
up to 10 .mu.m outside of the cell boundary. The mechanism of cell
death is due to the propagation of stress and shock waves in the
skin generated by the rapid deceleration of the microparticles
(McSloy et al. (2004)). The rapid rise time of these stress waves
in the skin, and their magnitude both contribute to cell death and
the results are consistent with the findings reported by Doukas, A.
G. & Flotte, T. J. (1996). Physical characterization and
biological effects of laser-induced stress waves. Ultrasound in
Medicine and Biology, 22(2):151-164. The effects of shock waves and
particle penetration in the skin on cell viability following gene
gun delivery. Masters Thesis, Department of Engineering Science,
University of Oxford.). This mechanism of ballistic particle
penetration killing cells negatively affects the ability of the
direct and efficient delivery of genes and drugs to the cells.
[0016] This cell death effect of ballistic particle delivery could
be reduced by significantly decreasing the particle size to the
nanometer regime-thereby reducing the stresses on the cells.
However, another limitation of the gene gun is that it is
unsuitable in delivering sub-micron sized particles to cells. This
is illustrated by the following. As reported (Kendall, M. A. F.,
Mitchell, T. J. & Wrighton-Smith, P. (2004) Intradermal
ballistic delivery of micro-particles into excised human skin for
drug and vaccine applications. J Biomechanics, 37(11):1733-1741),
and shown in FIG. 14, ballistic particle penetration is
proportional to the particle impact parameter, pvr, which is the
product of the particle density (p), velocity (v) and radius (r).
This parameter is also proportional to the particle momentum
per-unit-area, which has been shown to drive the mechanism of
particle penetration depth (Mitchell et al. (2003)). From FIG. 14,
we see a 1 .mu.m radius gold particle (density 18000 kg/m.sup.3)
would need to impact the skin at .about.600 m/s in order to
penetrate to reach cells .about.20 .mu.m into the skin.
[0017] Experimental results show that reducing the particle radius,
say, by an order of magnitude, to 100 nm, and placing it in a
standard biolistic device leads to negligible particle impact in
the skin. Indeed from FIG. 14 we see delivery to a 20 .mu.m depth
would need an impact velocity of .about.6000 m/s, which is
impractical for two reasons: 1) these hypervelocity conditions can
not be safely achieved with a system configured for human use (they
are usually achieved with massive free-piston shock tunnels); 2)
even if 6000 m/s was obtained in the free-jet, a gas impingement
region above the skin would seriously decrease the particle
velocity-it is possible that the particle would not even hit the
skin at all. Interestingly if a method was conceived to safely and
practically deliver nanoparticles to the skin at higher velocity
(e.g., the stated case of an 100 nm radius gold particle at a
velocity of .about.6000 m/s), the cell death benefit of smaller
scale would be offset by higher peak stresses-killing more
cells-and higher strain rates that are likely to further "toughen"
the skin, making delivery even more difficult.
[0018] In conclusion, these collective facts rule out the gene gun
as a viable option for delivering nanoparticles and therefore
precludes it from many of the developments in biomolecules, drugs
and sensors at this scale.
[0019] The huge research effort in micro- and nanotechnologies
provides tremendous potential for simple and practical cell
targeting strategies to overcome many limitations of current
biolistic (and other) cell targeting approaches. For example, FIG.
11(c) shows that the most conceptually simple and appealing
approach to gene delivery is the direct injection of naked DNA to
live cell nuclei at a sub-micrometer scale that does not adversely
damage the cell (Luo, D. & Saltzman, W. M. (2000) Synthetic DNA
delivery systems. Nature Biotechnology, 18:33-36). Cell death is
minimized by both the sub-micrometer scale of the injector and the
low, quasi-static strain-rate of the probe (compared to ballistic
delivery) resulting in low stress distributions. Although this is a
very efficient gene and bioagent delivery route, the to drawback is
that such precise targeting by direct microinjection can only be
achieved one cell at a time and with great difficulty to the
operator in vivo. Hence, the method is slow, laborious and
impractical.
[0020] Researchers have overcome some of these disadvantages for
transdermal drug delivery by fabricating arrays of micrometer-scale
projections (thousands on a patch) to breach the stratum corneum
for the intradermal delivery of antigens and adjuvants to humans
and other mammals.
[0021] In the scientific literature, the first description of this
technique appears to be the paper Microfabricated Microneedles: A
Novel Approach to Transdermal Drug Delivery. S. Henry et al, J.
Pharmaceutical Sci. vol. 87(8) p 922-925 (1998), with the
accompanying patent of U.S. Pat. No. 6,503,231. The objective of
U.S. Pat. No. 6,503,231 is to provide a microneedle array device
for relatively painless, controlled, safe, convenient transdermal
delivery of a variety of drugs and for biosampling. This is
achieved by the microneedles breaching the tissue barrier (e.g.,
for skin: the stratum corneum) and then the therapeutic or
diagnostic material is injected through the hollow microneedles
into the tissue. Specifically, in claim 1 of U.S. Pat. No.
6,503,231, it is stated that the microneedles are to be hollow,
with a length of 100 .mu.m-1 mm, and claim 3 states the width of 1
.mu.m-100 .mu.m, with subsequent claims stating ways the hollow
needles can be connected to reservoirs for the injection of
liquids, fabrication methods, materials and examples of drugs to be
delivered. Thus, U.S. Pat. No. 6,503,231 describes a patch suitable
for delivering materials and/or energy across tissue barriers. The
microneedles are hollow and/or porous to permit drug delivery at
clinically relevant rates across skin or other tissue barriers,
without damage, pain, or irritation to the tissue.
[0022] Other related microneedle devices in the patent literature
are U.S. Pat. Nos. 5,527,288 and 5,611,806. More recently published
patent applications on this topic are WO02/085446, WO02/085447,
WO03/048031, WO03/053258 and WO02/100476A2.
[0023] These microneedles array patch technologies have achieved
only limited success to date. Generally, there are a range of
approaches configured to breach the stratum corneum to allow an
enhanced take-up of drug in the viable epidermis. Although this has
not been discussed in the patents referred to above, based upon
reported research on ballistic particle delivery and cell death,
the low strain rate of application, combined with the cases of
smaller projections are likely to induce a lower incidence of cell
death near the tips, than ballistic microparticle to delivery.
Also, unlike ballistic microparticle delivery, these projections
are removed from the tissue-alleviating the possibility of adverse
effects of "carrier" materials delivered to the body, long
term.
[0024] However, unlike biolistic targeting (FIG. 11(b)), and the
direct injection of cells (FIG. 11(c)), these microneedle arrays do
not have the advantage of readily and directly targeting inside the
skin cells. This cellular/organelle targeting capability is key in
a range of existing and potential methods of vaccination, gene
therapy, cancer treatment and immunotherapy (Needle-free epidermal
powder immunization. Chen et al, Expert Rev. Vaccines 1(3) p265-276
(2002)) and diagnostic technologies.
[0025] Whilst U.S. Pat. No. 5,457,041 describes a patch for
targeting cells, this is only suitable for use in vitro, and
requires specialized apparatus to direct the micro-needles towards
identified cells. The apparatus uses a microscope, to allow an
operator to locate the cells in the sample tissue, and then direct
the application of the micro-needles appropriately. As a result,
this makes the device unsuitable for use in clinical environments,
and limits the ability of the device to elicit a desired biological
response.
[0026] Therefore, there still remains a need to provide
projection-based technology which achieves a more accurately
directed delivery of the active agent or stimulus to the desired
site of action surrounding or within cells, without appreciable
damage to them.
BRIEF SUMMARY
[0027] In a first broad form the present invention provides a
device for delivery of material or stimulus to targets within a
body to produce a desired response, the targets being at least one
of cells of interest, cell organelles of interest and cell nuclei
of interest, the device including a number of projections for
penetrating a body surface, and wherein:
[0028] a) the number of projections is selected to produce a
desired response, the number being at least 500; and,
[0029] b) a spacing between projections is at least partially
determined based on an arrangement of the targets within the body.
Typically the number of projections is selected by:
[0030] a) determining a likelihood of a projection targeting at
least one of the targets;
[0031] b) determining a number of targets to be targeted; and,
[0032] c) determining the number of projections using the
determined likelihood and the determined number of targets.
[0033] Typically the likelihood P.sub.contact of a projection
targeting a target of interest is at least partially based on:
P contact = V tar V layer ##EQU00001##
[0034] ii) where: [0035] (a) V.sub.layer is the volume of the layer
containing targets, [0036] (b) V.sub.tar is the volume including
the target of interest to which material or stimulus can be
delivered.
[0037] Typically the number of targets to be targeted depends on
the number of targets that need to be transfected to produce the
desired response.
[0038] Typically the number of targets to be targeted is at least
one of:
[0039] a) at least 10;
[0040] b) at least 100;
[0041] c) at least 1000;
[0042] d) at least 10000;
[0043] e) at least 100000;
[0044] f) at least 1000000; and,
[0045] g) at least 10000000.
[0046] Typically the number of projections is at least one of:
[0047] a) at least 10;
[0048] b) at least 100;
[0049] c) at least 1000;
[0050] d) at least 10000;
[0051] e) at least 100000;
[0052] f) at least 1000000; and,
[0053] g) at least 10000000.
[0054] Typically a maximum number of projections is based on at
least one of:
[0055] a) the total surface area of the target site available;
[0056] b) a minimum projection spacing (S); and,
[0057] c) an upper limit in active material or stimulus to be
delivered
[0058] Typically the projection spacing is based at least partially
on at least one of:
[0059] a) a size of the targets of interest; and,
[0060] b) a spacing between the targets of interest.
[0061] Typically the spacing between at least some of the
projections is selected to avoid multiple projections targeting a
single target of interest.
[0062] Typically the spacing between at least some of the
projections is selected to be greater than a diameter of the
targets of interest.
[0063] Typically the spacing between at least some of the
projections is selected to be approximately equal to the spacing
between the targets of interest.
[0064] Typically the spacing S between at least one of:
[0065] a) 1 .mu.m.ltoreq.S.ltoreq.10000 .mu.m; and,
[0066] b) 10 .mu.m.ltoreq.S.ltoreq.200 .mu.m.
[0067] Typically projection dimensions are based at least partially
on an arrangement of targets within the body.
[0068] Typically at least some of the projections have a diameter
of at least one of:
[0069] a) less than the size of targets; and,
[0070] b) of the order of the size of targets within the
targets.
[0071] Typically at least some of the projections have a projection
length at least partially based on a depth of the targets below a
surface of the body against which the device is to be applied in
use.
[0072] Typically the projections include a support section and a
targeting section.
[0073] Typically the targeting section has a diameter of less than
at least one of:
[0074] a) 1.mu.m; and,
[0075] b) 0.5 um.
[0076] Typically a length for the targeting section is at
least:
[0077] a) less than 0.5 um; and,
[0078] b) less than 1.0 um; and,
[0079] c) less than 2.0 um.
[0080] Typically a length for the support section is at least
partially based on a depth of the targets below a surface of the
body against which the device is to be applied in use.
[0081] Typically the length for the support section is at least
partially determined in accordance with properties of a surface of
the body against which the device is to be applied in use.
[0082] Typically at least one of a support section length and the
number of projections is at least partially based on a likelihood
of a projection penetrating the targets:
P d .times. e .times. p .times. t .times. h = .intg. B - Q .times.
.sigma. T + B - Q .times. .sigma. .times. 1 .sigma. .times. 2
.times. .pi. .times. e - ( x - D .sigma. ) 2 ##EQU00002##
where:
[0083] (a) .sigma. is the standard deviation from a mean location,
accounting for the skin surface undulations.
[0084] (b) D is a distance of the targets below a surface of the
body against which the device is to be applied in use;
[0085] (c) Q is a number of standard deviations from a mean level
of the surface of the body at which the device comes to rest in
use;
[0086] (d) B is a length of the support section; and,
[0087] (e) T is a length of a targeting section.
[0088] Typically a length for the support section is at least one
of:
[0089] a) for epidermal delivery <200 .mu.m;
[0090] b) for dermal cell delivery <1000 .mu.m;
[0091] c) for delivery to basal cells in the epithelium of the
mucosa 600-800 .mu.m; and,
[0092] d) for lung delivery of the order of 100 .mu.m in this
case.
[0093] Typically the length of the delivery end section is greater
than the target dimension.
[0094] Typically at least some of the projections within a
targeting configuration have different dimensions.
[0095] Typically the projections are solid.
[0096] Typically the projections are non-porous and non-hollow.
[0097] Typically at least part of at least some of the projections
are coated with a bioactive material.
[0098] Typically at least part of at least some of the projections
are coated with a non-liquid material.
[0099] Typically at least part of a targeting section of at least
some of the projections are coated.
[0100] Typically the coating is at least one of:
[0101] a) nanoparticles;
[0102] b) a nucleic acid or protein;
[0103] c) an antigen, allergen, or adjuvant;
[0104] d) parasites, bacteria, viruses, or virus-like
particles;
[0105] e) quantum dots, SERS tags, raman tags or other
nanobiosensors;
[0106] f) metals or metallic compounds; and,
[0107] g) molecules, elements or compounds.
[0108] Typically the device includes at least some uncoated
projections to thereby stimulate or perturb the targets in use.
[0109] In one example, the device includes:
[0110] a) a flexible substrate; and,
[0111] b) a number of patches, each patch including a number of
projections for penetrating a body surface, the number of patches
being mounted to a flexible backing.
[0112] In a second broad form the present invention provides a
method of selecting constructional features for a device a for
delivery of material or stimulus to targets within a body to
produce to a desired response, the targets being at least one of
cells of interest, cell organelles of interest and cell nuclei of
interest, the device including a number of projections for
penetrating a body surface, and wherein the method includes:
[0113] a) selecting the number of projections to produce a desired
response, the number being at least 500; and,
[0114] b) selecting a spacing between projections at least
partially based on an arrangement of the targets within the
body.
[0115] In a third broad form the present invention provides a
method of fabricating a device for delivery of material or stimulus
to targets within a body to produce a desired response, the targets
being at least one of cells of interest, cell organelles of
interest and cell nuclei of interest, the device including a number
of projections for penetrating a body surface, and wherein the
method includes:
[0116] a) selecting the number of projections to produce a desired
response, the number being at least 500;
[0117] b) selecting a spacing between projections at least
partially based on an arrangement of the targets within the body;
and,
[0118] c) fabricating the device using the selected number of
projections, and the selected spacing
[0119] In a fourth broad form the present invention provides a
method of treating a subject the method including using a device
for delivery of material or stimulus to targets within the
subject's body to produce a desired response, the targets being at
least one of cells of interest, cell organelles of interest and
cell nuclei of interest, the device including a number of
projections for penetrating a body surface, and wherein:
[0120] a) the number of projections is selected to produce a
desired response, the number being at least 500; and,
[0121] b) a spacing between projections is at least partially
determined based on an arrangement of the targets within the
body.
[0122] In a fifth broad form the present invention provides
apparatus for delivery of material or stimulus to targets of
interest within a body to produce a desired response, the targets
being at least one of cells of interest, cell organelles of
interest and cell nuclei of interest, the apparatus including:
[0123] a) a structure;
[0124] b) a plurality of projections movably mounted to the
structure for penetrating a body surface;
[0125] c) an actuator for selectively releasing the plurality of
projections mounted on the movable structure from a retracted
position such that upon contact with the body surface the plurality
of projections enter the body.
[0126] Typically the plurality of projections are provided on a
patch.
[0127] Typically the actuator includes:
[0128] a) a spring coupled to the structure and the at least one
patch; and,
[0129] b) a releasing means for releasing the spring, to thereby
release the plurality of projections from the retracted
position.
[0130] Typically the releasing means is a tensioned string for
holding the spring in compression.
[0131] Typically the releasing means is manually operated.
[0132] Typically the apparatus includes a number of arms, each arm
being coupled to a respective spring and including a first end
pivotally mounted to the structure and a second end coupled to a
respective plurality of projections, and wherein activation of the
releasing means causes each of the arms to be released from a
retracted position to thereby cause projections on the respective
patch to enter the body.
[0133] Typically the arms are circumferentially spaced around a
part of the structure.
[0134] Typically the structure is flexible structure allowing the
structure to be guided to a desired location within the body.
[0135] Typically the releasing means includes an inflatable
structure coated with the plurality of projections.
[0136] Typically:
[0137] a) the number of projections is selected to produce a
desired response, the number being at least 500; and,
[0138] b) a spacing between projections is at least partially
determined based on an arrangement of the targets within the
body.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0139] An example of the present invention will now be described
with reference to the accompanying drawings, in which:
[0140] FIGS. 1A and 1B are schematic diagrams of an example of
device for delivery of material or stimulus to targets within a
body;
[0141] FIG. 1C is a schematic diagram of an example of the device
of FIG. 1A in use;
[0142] FIGS. 1D and 1E are schematic diagrams of examples of
projections used in the device of FIG. 1A;
[0143] FIG. 2 is a flow chart of an example of the process of
selecting device parameters;
[0144] FIGS. 3A and 3B are schematic diagrams of alternative
examples of the device of FIG. 1A in use taking into account
variations in surface properties and target locations;
[0145] FIG. 4 is a flow chart of a second example of the process of
selecting device parameters;
[0146] FIGS. 5A and 5B show examples of the relationship between
the number of projections and total hits for targeting Langerhans
cell nuclei and Langerhans cells respectively;
[0147] FIG. 6 shows an example of the relationship between the
total number of targeted LC and the patch surface area as a
function of projection spacing geometry;
[0148] FIG. 7 is an SEM photograph of an example of a constructed
patch;
[0149] FIG. 8 shows an example of the Transfection Probability vs
Needle Spacing, for targeting of Langerhans cells;
[0150] FIGS. 9A to 9C show examples of projection viability against
the projection height, for variation in the skin surface level
standard deviation of 20 .mu.m, 40 .mu.m and 60 .mu.m
respectively;
[0151] FIG. 10 is a schematic diagram of an alternative example of
a patch;
[0152] FIG. 11 illustrates a schematic cross-section of skin
structure: (a) half-section scale of a typical smaller needle and
syringe (diameter .about.0.5 mm); (b) penetration of microparticles
following biolistic delivery; (c) idealized direct injection of a
cell nucleus; (d) a micro-nanoprojection array;
[0153] FIGS. 12A and 12B illustrate the effects of relative
humidity (A) and ambient temperature (B) on to ballistic particle
penetration into the skin;
[0154] FIG. 13A is a graph showing the relationship between
percentage cell death (membrane rupture) and particle density
(McSloy (2004) MA Thesis, University of Oxford);
[0155] FIG. 13B is a diagram showing how the data for FIG. 13A was
retrieved (McSloy (2004) MA Thesis, University of Oxford);
[0156] FIG. 13C is a graph showing membrane rupture versus distance
of cell pathway;
[0157] FIG. 14 illustrates the particle penetration parameter (pvr)
vs. penetration depth obtained by the ballistic delivery of gold
microparticles into skin (Kendall et al. (2004), Journal of
Biomechanics);
[0158] FIG. 15 shows examples of organelles within the cell
(http://niko.unl.edu/bs101/notes/chapter4.html);
[0159] FIG. 16 is a schematic diagram of the skin structure;
[0160] FIG. 17 is an example of a histology micrograph of human
skin with a Langerhans Cell (L) and Keratinocyte (K) stained. From
Roitt et al, the height of which is approximately 50 .mu.m;
[0161] FIG. 18 illustrates the distribution of Langerhans Cells in
a mouse ear (Kendall M. A. F., Mulholland W. J., Tirlapur U. K.,
Arbuthnott E. S., and Armitage, M. (2003) "Targeted delivery of
micro-particles to epithelial cells for immunotherapy and vaccines:
an experimental and probabilistic study", The 6th International
Conference on Cellular Engineering, Sydney, August 20-22);
[0162] FIGS. 19A and 19B illustrate (A) a sample of canine buccal
mucosal tissue and (B) the structure of the mucosa, Mitchell, T
(2003) DPhil Thesis, Department of Engineering Science, University
of Oxford;
[0163] FIG. 20 illustrates the shape and dimensions of examples
nanoneedles;
[0164] FIG. 21 illustrates the maximum needle length vs. nanoneedle
diameter calculated using expression (3) and the Young's Modulus
(E) values for Gold, Titanium, ZnO, PGCA, Silver and Tungsten
(respectively: 77.2, 116, 111.2 and 7 GPa);
[0165] FIGS. 22A-22C are a Transmission Electron Micrographs (TEM)
of a micro-nanoprojection electropolished from tungsten at (A)
.times.500 magnification, (B) a bright field .times.33000
magnification and (C) a dark field .times.33000 magnification;
[0166] FIGS. 23A and 23B illustrate fluorescent microscope images
of tungsten rods, (A) uncoated and (B) coated in eGFP plasmid DNA
immersed in an ethidium bromide solution;
[0167] FIGS. 24A-24D are examples of an optically sectioned
Multi-Photon Microscopy (MPM) images of the agar after insertion of
a DNA coated tungsten probe (A) on the surface (B) at a depth of 13
.mu.m (C) at a depth of (32 .mu.m). Also shown in (D) is an optical
section at 32 .mu.m of agar gel following insertion of a probe
without a DNA coating;
[0168] FIG. 25 illustrates a photomicrograph of a
micro-nanoprojection electropolished from tungsten used in skin
tissue indentation experiment with scale bar;
[0169] FIG. 26 illustrates two sample load-displacement curves in
freshly excised mouse ear tissue obtained with the
micro-nanoprojection shown in FIG. 25;
[0170] FIG. 27 is a plan view diagram of possible alternative
geometry of the nanoneedle;
[0171] FIG. 28 illustrates an example of a nanoneedle array or
patch device;
[0172] FIG. 29 is a schematic diagram of an example of the
nanoneedle array produced with 2PLSM;
[0173] FIGS. 30A-30C illustrate sequences for producing a mask;
[0174] FIG. 31 is a schematic diagram of an example of a "Stepped"
nanoneedle;
[0175] FIGS. 32A and 32B illustrate examples of an intradermal
application of nanoneedle patches;
[0176] FIG. 33 is a schematic diagram of an example of an
applicator, fitted with the patch for mucosal delivery;
[0177] FIG. 34 illustrates the major respiratory organs; and,
[0178] FIGS. 35A-35C are schematic diagrams of an example of a
deployable patch structure for targeting the lower airway and
lung.
DETAILED DESCRIPTION
[0179] An example of a device for delivering material or stimulus
targets within a body will now be described with reference to FIG.
1A to 1E.
[0180] In this example, the device is in the form of patch 100
having a base 120 and a number of projections 110. The base 120 and
projections 110 may be formed from any suitable material, as will
be described in more detail below, but in one example, are formed
from a silicon type material, allowing the device to be fabricated
using fabrication processes such as vapor deposition, silicon
etching, Deep Reactive Ion Etching (DRIE), or the like.
[0181] In the example shown, the device has a width W and a breadth
B with the projections 110 being separated by spacing S.
[0182] In use, the patch 100 is positioned against a surface of a
subject, allowing the projections to enter the surface and provide
stimulus or material to targets therein. An example of this is
shown in FIG. 1C.
[0183] In this example, the patch 100 is urged against a subject's
skin shown generally at 150, so that the projections 110 pierce the
Stratum Corneum 160, and enter the Viable Epidermis 170 to reach
targets of interest, shown generally at 180.
[0184] Examples of suitable projections are shown in more detail in
FIGS. 1D and 1E.
[0185] In each example, the projection generally includes a
targeting section 111, intended to deliver the material or stimulus
to targets within the body, and a support section 112 for
supporting the targeting section 111.
[0186] In the example of FIG. 1D, the projection is formed from a
conically shaped member, which tapers gradually along its entire
length. In this example, the targeting section 111 is therefore
defined to be the part of the projection having a diameter of less
than d.sub.2.
[0187] As an alternative example however, the structure of the
projection may vary along its length to provide a targeting section
111 with a designed structure. In this example, the targeting
section 111 is in the form of a substantially cylindrical shape,
such that the diameter d.sub.1 is approximately equal to the
diameter d.sub.2. In either case, the support section has a length
a, whilst the targeting section 111 has a length l. The diameter of
the tip is indicated by d.sub.1, whilst the diameter of the support
section base is given by d.sub.3.
[0188] In use, the device is intended to deliver material or
stimulus to specific targets within the body. Thus, rather than
just operating to deliver material to, for example, the blood
supply, or tissue within the body, the device is configured so as
to ensure material or stimulus reaches specifically selected
targets such as cells, cell organelles, cell nuclei, or the like.
Furthermore, the device is designed to achieve this without
requiring specific directional control of device application so as
to ensure the projections reach the targets. In other words, the
device is intended to ensure successful delivery of material or
stimulus to specific targets within a subject, without requiring
that the projections are aimed at the specific targets, but rather
by allowing general placement in a suitable region. Thus, for
example placement may to be as simple as placement anywhere on the
user's skin in order to target Langerhans cells of the device on
the subject.
[0189] To achieve this, the device is provided with a particular
configuration to ensure successful targeting. Accordingly, it is
generally necessary to select patch parameters, such as the number
of projections N, and spacing S between projections, to be
dependent upon the intended use of the device. A mechanism for
achieving this will now be described with reference to FIG. 2.
[0190] In this example, at step 200 it is necessary to determine an
arrangement of desired targets. This may be achieved in any one of
a number of ways and will depend on the nature of the targets.
Thus, for example if the targets are a specific type of cell, cell
nuclei, or cell organelle, this information can be determined from
literature or studies detailing the typical location of cells, or
other targets, within the body.
[0191] At step 210 a number of projections required to elicit a
desired response is determined. This can depend on a range of
factors, such as the ability of projections to reach the desired
targets, the ability of the projections to deliver material or
stimulus to the targets, as well as the ability of the targets to
elicit a response. Thus, for example, non-uniform distribution of
targets within the body means that it is not possible to assume
that each projection will deliver material or stimulus to a desired
target during use of the device.
[0192] The number of projections may be determined in any one of a
number of ways. Thus, for example, this can include selecting a
number of projections from a predetermined list outlining the
number of projections required for specific uses. However, if the
number has not previously been determined, for example, if the
target has not previously been used, then some form of analysis is
typically required.
[0193] In one example, this is achieved by analyzing the
distribution of targets within the body and then determining a
likelihood of any one projection reaching a target. An indication
of the number of targets to which stimulus or material must be
delivered can then be used to determine an indication of the number
of projections required.
[0194] As will be described in more detail below, in general a
desired response cannot be obtained with less than 500 projections.
More typically at least 750 projections are required. However, in
some instances, even more projections such as at least 1000, 2000,
5000, 7500, 10,000, 100,000, 1,000,000 or even 10,000,000 may be
used, and specific examples will be described in more detail
below.
[0195] Once a number of projections has been selected, a projection
spacing is determined at least partially based on the target
arrangement at step 230.
[0196] The spacing may be determined in any one of a number of
ways, but typically includes setting a lower spacing limit to
ensure that only a single projection delivers material or stimulus
to a single target. The maximum spacing S is typically set based on
the required patch size (B.times.W) and/or the spacing between
targets. It will be appreciated that whilst the example shown is
rectangular, alternative shapes, such as circular, elliptical,
hexagonal, or the like, may be used and that the use of a
rectangular patch is for the purpose of example only.
[0197] At step 240 a patch is fabricated in accordance with the
selected patch parameters, including the number of projections N
and the spacing S. Fabrication may be achieved in a number of ways,
as will be described in more detail below.
[0198] By selecting at least a number of projections N required to
elicit a desired response, this allows a patch to be provided with
sufficient projections to ensure that a desired response is
achieved by delivery of material or stimulus to specific targets.
Furthermore, by utilizing a probabilistic analysis, this technique
ensures that the required targeting will be achieved without
requiring the individual projections to be aimed at the individual
targets. Thus, in contrast to other prior art techniques, the patch
100 may simply be inserted into a body at a general location, and
does not need specialized apparatus to direct the projections
towards specific cells or other targets within the body.
[0199] A more detailed example of the process will now be
described. For the purpose of this example the patch configuration,
and in particular the insertion of the patch into the body is as
shown in FIG. 3A and FIG. 3B. In particular, this example is
modified to take into account variations and undulations in the
surface of the body, as well as variations in target depth.
[0200] In this example, the patch 100 is urged against the surface
300 of the Stratum Corneum 310. The surface 300 includes
undulations, resulting in a mean surface level 320 shown by dotted
to lines, with the patch base 120 resting against the surface 300
at a distance y above the mean level 320.
[0201] The projections 110 enter the Viable Epidermis 330 to
deliver material or stimulus to targets 340, which are generally
arranged in a layer 350, referred to as the target layer. The
Dermis is also shown at 360 in this example.
[0202] In the example of FIG. 3A the targets 340 are provided in a
single layer with each target being approximately a constant depth
D.sub.c below the Stratum Corneum 310. In this example, the layer
height h.sub.layer is therefore approximately equal to the diameter
of the targets d.sub.c, with the targets separated by a spacing
S.sub.c. It would be appreciated by persons skilled in the art that
in this instance the targets may therefore be Langerhans Cells, or
the like.
[0203] In the example of FIG. 3B, the targets 340 are dispersed
vertically through the Viable Epidermis 330, so that the target
layer 350 has a greater height h.sub.layer than in the previous
example. Additionally, in the example, the depth of the targets is
calculated on the basis of the mean layer depth, as shown.
[0204] An example of the process for selecting a device
configuration to take into account the arrangements in FIGS. 3A and
3B will now be described in more detail with reference to FIG.
4.
[0205] At step 400 a desired target and corresponding target
arrangement is determined.
[0206] The target selected will depend on the intended application.
Thus, for example, the target may be cells, cell nuclei or cell
organelles. Additionally, different types of cells may need to be
targeted. Thus, for example, cells such as Langerhans Cells may be
stimulated for providing an immunological response, whereas cells
such as squamous or basal cells may need to be targeted to treat
cell carcinoma. An example of other potential targets will be
described in more detail below.
[0207] Determining the target arrangement typically involves
determining parameters relating to the target such as target depth
D.sub.c, target diameter d.sub.c, layer height h.sub.layer and
target spacing S.sub.c. Thus these correspond to the parameters
outlined above with respect to FIGS. 3A and 3B.
[0208] At step 410 the diameter of the targeting section 111 for at
least some of the projections is determined.
[0209] The diameter of the targeting section is typically based on
the size of the target. Thus, for example, the diameter of the
targeting section does not usually exceed the scale of the target,
as this may lead to target necrosis. In general this leads to an
upper limit for targeting section diameters of:
d.sub.1.ltoreq.1 .mu.m and d.sub.2.ltoreq.2 .mu.m.
[0210] However, it will be appreciated that smaller diameters such
as 500 nm, or below, may be used, as described in specific examples
below. Additionally or alternatively, it may be desirable to
include projections having a larger diameter, for example to cause
cell necrosis. In one example, at least some of the projections
have a diameter greater than 1 82 m, which can be used to induce
bystander responses. In other examples, all of the projections have
a diameter greater than 1 82 m to thereby kill targets.
[0211] At step 420 the projection length is determined. In one
example, the projection length is based on the depth of the target
layer D.sub.c and the layer height h.sub.layer. Thus, the length of
the supporting section of a can be selected so that the targeting
section 111 at least reaches the target layer within the body, but
typically does not extend a large distance beyond the target layer.
In this example, the length a of the support section 112 is
typically given by:
(D.sub.c+h.sub.layer/2).gtoreq.a.gtoreq.(D.sub.c-h.sub.layer/2)
[0212] Similarly the length l of the targeting section 111 is
typically selected to be at least equal to the layer height
h.sub.layer to ensure penetration of targets within the layer 340,
so that:
l.gtoreq.h.sub.layer
[0213] As shown in the examples of FIGS. 3A and 3B however the skin
of the body is not generally flat but is undulating. As a result,
this means that the base 120 of the device 100 generally sits a
distance y above the mean skin level 320. To take this into
account, the length of the support section 112 may be increased
such that.
(y+D.sub.c+h.sub.layer/2).gtoreq.a.gtoreq.(y+D.sub.c-h.sub.layer/2)
[0214] Alternatively, a probabilistic analysis can be used to
determine the likelihood of a viable to projection of a given
length reaching the targets with the body. This will depend on a
number of factors, examples of which, for the targeting of
Langerhans cells (LC), include:
[0215] The surface of the skin is normally distributed
[0216] It is assumed that the LC reside exactly 17 82 m below the
surface just above the basal layer (Arbuthnott, 2003, Emislom et
al, 1995), in the case of the Balb/c mouse ear. In reality, there
may be a small variation in depth of LC. For example, the depth of
LC varies significantly from site to site within a given animal of
human model, and there is typically variation in the depth of LC
between models-for example the depth of LC in humans is greater
than in mice.
[0217] The patch typically comes to rest two standard deviations
away from the mean skin level
[0218] The needles have a viable tip length of 20 82 m (i.e., l,
(defined as 111) is 20 .mu.m. Penetration by any other part of the
needle other than this tip causes cell death.
[0219] The skin's surface is normally distributed with mean 0 and
standard deviation .sigma..
[0220] Within the model:
[0221] The Langerhans cells lie D.sub.c microns directly below the
skin's surface.
[0222] The patch comes to rest Q standard deviations from the
skin's mean level.
[0223] The needles have body (unviable) length support section a
and tip (viable) length l.
[0224] In this example, the likelihood of viable targeting of
targets at the defined depths (P.sub.depth) is given by:
P d .times. e .times. p .times. t .times. h = .intg. a - Q .times.
.sigma. l + a - Q .times. .sigma. .times. d .times. x .sigma.
.times. 2 .times. .pi. .times. e - ( x - D c .sigma. ) 2 ( 1 )
##EQU00003##
where:
[0225] .sigma. is the standard deviation from a mean location,
accounting for the skin surface undulations.
[0226] D.sub.c is a distance of the cells of interest below a
surface of the body against which the device is to be applied in
use;
[0227] Q is a number of standard deviations from a mean level of
the surface of the body at which the device comes to rest in
use;
[0228] a is a length of the support section; and,
[0229] l is a length of a targeting section.
[0230] In this example it is assumed that the skin surface level is
normally distributed on a local scale, which is typically a safe
assumption based on histology samples, although if the area
inspected is too large, global curvature invalidates the
assumption. At first instance, it is assumed that the patch comes
to rest at a distance y of two standard deviations away from mean
skin level.
[0231] The use of such a model allows support section and targeting
section lengths a, l to be selected to ensure a predetermined
probability of the targeting section 111 successfully reaching the
targets. In one example, the probability is selected to be one, to
ensure successful delivery of the material or stimulus.
Alternatively, lower probabilities may be selected, with this being
taken into account in determining the number of projections
provided, as will be described in more detail below.
[0232] At step 430, the likelihood of a single targeting event is
determined. In particular, this is the likelihood of a projection,
assuming the projection reaches the target layer 350, of delivering
material to or stimulus to a respective target 340.
[0233] In one example, it is assumed that the targeting section 111
is effectively a point diameter cylinder that extends into the
target layer 350. In this instance the likelihood of a single
targeting event is given by the volume of the target divided by the
volume of the layer.
P contact = V tar V layer ( 2 ) ##EQU00004##
where:
[0234] V.sub.layer is the volume of the layer containing cells of
interest,
[0235] V.sub.tar is the volume including the target to which
material or stimulus can be delivered.
[0236] In this regard it will be appreciated that the volume to
which stimulus or material may be to delivered may be significantly
greater than the size of the physical target itself, depending on
the delivery mechanism used. Thus, for example, if material to be
delivered to a cell is absorbed when placed within the vicinity of
the cell, then the target volume V.sub.tar will be larger than the
volume of the cell.
[0237] However, in a more detailed example, equation (2) still
holds, but it can be assumed that the projection has its own volume
(V.sub.pro), and in this case the probability of contact is
modified to change V.sub.tar to take into account the additional
probe volume. In one example, if the projection is as shown in FIG.
1E, in which d.sub.1=d.sub.2, the volume of the targeting section
within the target layer is given by:
V tar = i = 1 N tar .times. ( ( 2 .times. d 1 + d c ) 2 .times. d c
) ( 3 ) ##EQU00005##
where:
[0238] N.sub.tar is the number of targets, with diameter
d.sub.c.
[0239] Equation (3) can be modified to take into account variations
in target size and shape, and probe size and shape. When d.sub.1
and d.sub.c are constant, equation (3) becomes:
V.sub.tar=N.sub.Tar(2d.sub.1+d.sub.c).sup.2d.sub.c (4)
[0240] So for a region of target layer of size z.times.z, where a
projection reaches through the target layer, and containing a
number of targets N.sub.tar, the probability is given by:
P contact = N Tar .function. ( 2 .times. d 1 + d c ) 2 .times. d c
z 2 .times. h layer ( 5 ) ##EQU00006##
[0241] The above example assumes that the projection reaches fully
through the target layer 350 (i.e., P.sub.depth=1). However, this
may not occur, and accordingly the above described equation can be
utilized to take into account the probability of the projection
reaching the relevant cell layer, as described in equation (1)
above.
[0242] So the general expression for the probability of a
projection targeting the targets of interest (e.g., key cells,
nuclei, etc.), defined as P.sub.tar is:
P.sub.tar=P.sub.contact.times.P.sub.depth (6)
[0243] It will be appreciated that in the event that the projection
lengths a, l are selected such that P.sub.depth=1, then
P.sub.tar=P.sub.contact.
[0244] At step 440 the number of targeting events required is
determined. This is typically determined on the basis of studies
performed indicating the number of targets to which material or
stimuli must be delivered in order for a biological response to be
affected. Often, this is determined by parametric empirical
studies.
[0245] For example, when it is desired to deliver DNA to transfect
Langerhans cells, at least one cell must be targeted. However, it
is believed that to ensure a successful biological response, at
least 10 cells, and more preferably at least 100 cells and up to or
over 1,000 cells are targeted. It will be appreciated however that
for different delivery mechanisms a different number of targets may
be desirable.
[0246] At step 450, having determined the number of targeting
events required (N), it is possible to use this and the probability
of a single targeting event to determine a number of projections
required, which is given by:
N=T/P.sub.tar
where:
[0247] T is the total number of delivery events required to produce
the desired response.
[0248] Often in immunotherapeutic and drug applications (including
vaccines), T is defined as a range, that can vary significantly
between application and/or model (i.e., animal or human).
[0249] Specific examples of this are shown in FIGS. 5A and 5B, for
the targeting of Langerhans cell nuclei and Langerhans cells
respectively.
[0250] In these examples it is assumed that:
[0251] A Langerhans Cell (LC) penetrated by one needle remains
viable. If it is penetrated by any more than one projection cell
death occurs.
[0252] When a projection contacts the target site (e.g., cell
nucleus or cytosol), the desired biological "event" occurs each
time. In reality, there is probability of the event happening. For
example, if cell transfection is required, then the probability of
this event through the delivery of DNA to nucleus via a projection
is .about.0.9, whereas the probability of the same event from the
coated projection entering only the cytoplasm is .about.0.1
(Nagasaki 2005).
[0253] A dead cell cannot be transfected.
[0254] Penetration by a needle with a diameter greater than 1 .mu.m
will cause cell death.
[0255] LC are assumed as spherical with 10 .mu.m diameter.
[0256] Nuclei of LC are assumed to be spherical, with a diameter of
4 82 m (Arbuthnott, 2003).
[0257] All LC lie just above the basal layer between the epidermis
and dermis (as reported by Kendall et al. (2003).
[0258] LC are oval with dimensions circa 11.25.times.6 .mu.m but to
simplify the model, the average of these two figures is taken as
the diameter of 10 .mu.m. In one example of young Balb/C mice the
LC density is 895 cells/mm.sup.2 (Choi. et al 1987). With LC
uniformly distributed in the suprabasal region (Numahara et al.
2001) this gives a center to center spacing of approximately 30
microns, justifying the spacing assumption. For simplicity, it is
assumed that the LC spacing of 1000 cells/mm.sup.2, giving a planar
spacing between cell centers of 32 .mu.m.
[0259] FIG. 5A shows the relationship between the number of
projections and total hits is given for the patches 100 described
above, with a range of depth probabilities (P.sub.depth), for
targeting Langerhans cell nuclei.
[0260] FIG. 5B shows the relationship between the number of
projections and total hits is given for the patches 100 described
above, with a range of depth probabilities (i.e., the probability
of a viable targeting of the target layer 350), for targeting
Langerhans cells.
[0261] Typically this requires at least 500 projections and more
typically at least 1,000 projections for targeting LC and 10,000
projections for targeting LC nuclei. Specific examples of this will
be described in more detail below.
[0262] At step 460 the projection spacing (S) is then determined
based on the target diameter and target spacing. It is usual to
assume that no more than one projection should enter a single cell
this; will typically lead to cell death. Accordingly, it is typical
to select a projection spacing which is at least greater than a
cell diameter, such that:
S.gtoreq.d.sub.c
[0263] More preferably it is typical to select a size for the
projection spacing based on a preferred overall patch size. In
particular, it is preferred that patches are made below a certain
upper limit defined by practical utility to the targeting site of
the patient or animal.
[0264] For example, if the targeting site is the skin of the human
abdomen, then the surface area could be approaching the surface
area of the abdomen (e.g., .about.400 cm.sup.2, or 20.times.20 cm,
say). It would not be practical to achieve such larger surface
areas over surfaces with "bulk" curvature (such as the abdomen
example, or arm) with one large rigid patch. In one example,
surface area can be achieved using the patch shown in FIG. 10. In
this example, the patch 100 is formed from several smaller patches
1000 provided on a flexible backing material 1010. The patch
assembly could be extended to wrap around the patient site
1020.
[0265] In any event, an upper limit on the spacing S is typically
selected to ensure that the desired number of projections fit on a
patch of the desired size.
[0266] A specific example of this is shown in FIG. 6, in which the
relationship between the total number of targeted LC (T) and the
patch surface area (mm.sup.2) as a function of spacing geometry, is
shown. In this example, the probability of penetration to the cell
depth is assumed to be one (i.e., P.sub.depth=1) for
simplicity.
[0267] Irrespective of this, the projection spacing S is typically
of the order of spacing of the targets S.sub.c.
[0268] Once the spacing is selected the patch can be fabricated at
step 470. In general, this will typically require considerations
are taken into account to ensure the projections are sufficiently
robust to withstand penetration of the Stratum Corneum and Viable
Epidermis without breaking. Example manufacturing processes will be
described in more detail below, and an example of a constructed
patch 100 is shown in FIG. 7. In this example, the device has 9795
projections, with a spacing of 70 82 m (S), over a surface area of
48 mm.sup.2.
[0269] In general, a number of factors regarding the patch
fabrication should be noted.
[0270] Typically the projections are solid, non-porous and
non-hollow. The use of solid projections enhances the projection
strength, thereby reducing the likelihood of projection breakage,
which in turn helps maximize successful delivery of material or
stimulus to the targets. Also, solid projections simplify device
fabrication processes, allowing for the production of cheaper
patches than for the case of porous or hollow projections. This in
turn further enhances the suitability of the patch for use in
medical environments.
[0271] To achieve delivery of material, it is typical to coat at
least the targeting section 111 with a non-liquid bioactive
material, such as DNA.
[0272] The patch 100 may also be fabricated to perturb targets so
as to induce "bystander" interactions. This may be used, for
example, so that cell death is used to release molecules to
activate nearby targeted cells. This can be achieved in a number of
manners, such as by providing a mixture of coated and uncoated
projections, as well as by providing projections of differing
dimensions including clusters of more than 1 projection to target
individual cells and/or larger scale tips to damage cells or other
targets, upon insertion/residence/retrieval.
SPECIFIC EXAMPLES
[0273] A number of specific examples will now be described.
[0274] Transfection Probability
[0275] In this example, which focuses on the transfection of
Langerhans cells, a number of additional practical considerations
may also be taken into account.
[0276] In particular, if the model of a point targeting section
with no radius is used, this predicts an idealized projection
spacing S=6.5 .mu.m, as used in the example of FIG. 8. In this
case, the LC diameter is .about.10 .mu.m, so that to satisfy the
requirements set out above, namely that S.gtoreq.d.sub.c, so
S.gtoreq.10 .mu.m.
[0277] Also, for structural reasons, the diameter of the base of
the projections d.sub.3 is likely to be above 6.5 .mu.m, and a
"clearance" will be needed between each projection, so for
practical reasons, the minimum projection spacing (S) is at least
10 .mu.m.
[0278] FIG. 8 shows an example of the Transfection Probability vs
Needle Spacing, for targeting of Langerhans cells with a spacing of
32 .mu.m. The S.gtoreq.d.sub.c criterion and practical
considerations of minimum size of the base d.sub.3 suggest the
spacing is to at least be 10 .mu.m, as discussed above.
[0279] Needle Length Optimization
[0280] For this example, the sensitivity of needle length to
standard deviation of the skin surface is shown in FIG. 9A to 9C.
In particular, these show the fraction of projections (P.sub.depth)
that are smaller than 1 .mu.m in diameter and penetrate to 17 82 m
in the epidermis (the depth of LC), against the projection height,
for skin level standard deviation of 20 82 m, 40 .mu.m and 60 82 m
respectively.
[0281] It is clear from this, that the magnitude of skin
undulations has a strong influence over the choice of optimum
projection length.
[0282] Minimum Number of Projections
[0283] In one example, for targeting a single LC nucleus the
likelihood of contact of a projection is given by
P.sub.contact=0.031, using equation (2) above, and data regarding
LC nuclei size, set out in more detail below. The value of
P.sub.depth defined in equation (1), above has a range of 0-1, and
with N=500, P.sub.depth=0.064.
[0284] Utilizing this, it can be seen that a viable range of
lengths result in targeting of a single cell, as shown below.
TABLE-US-00001 SD Needle (roughness) length needed FIG. (.mu.m) (T
= 1; N = 500) 9A 20 >80 .mu.m. 9B 40 >130 .mu.m. 9C 60
>160 .mu.m
[0285] All of these cases in the table above are well away from the
optimal lengths for the projections (i.e., where P.sub.depth is a
maximum in FIGS. 9A-9C), so they do represent a "poor" case
scenario, where the device is not tuned well for the target (device
tuning would be improved by reducing the needle length to closer to
the optimal regions shown in FIG. 9A-9C, for example). However, the
target of LC nuclei is very well defined. So, on balance, a minimum
of 500 projections is needed before there is a reasonable
statistical chance of just one targeting event.
[0286] It will be appreciated that it is typical however to deliver
to more than one target, and accordingly, it is typical for a
greater number of projections to be provided.
[0287] For example, the direct targeting of 2 LC nuclei (i.e., T=2)
would be achieved with the device configurations in the table above
with 1000 projections (i.e., N=1000).
[0288] Similarly, the direct targeting of 10 LC nuclei (i.e., T=10)
would be achieved with the device configurations in the table above
with 5000 projections (i.e., N=5000).
[0289] In the case of targeting situations with a lower
P.sub.contact, for instance in targeting a sparsely-populated
dermal dendritic cell phenotype (not in a tightly defined layer
like LC), greater than the minimum of 500 projections would be
needed for at least one single targeting event. For example, if
P.sub.contact is 1/10 of the stated LC case (i.e.,
P.sub.contact=0.0031), then applying a similar analysis, and
adjusting for the deeper location of cells to maintain
P.sub.depth=0.064, then 5000 projections would be needed for the
one targeting event.
[0290] Again, it will be appreciated that it is typical however to
deliver to more than one target, and accordingly, it is typical for
a greater number of projections to be provided.
[0291] Maximum Number of Projections
[0292] The upper limit of projections is typically defined by a
range of parameters. These include the total surface area of the
target site available, and the minimum projection spacing (S). For
example, in previously stated case of a human abdomen, with the
patch assembly wrapped around to the back (i.e., .about.800
cm.sup.2), and the minimum spacing for targeting cells (S=10 .mu.m)
results in 8,000,000 projections. Another consideration is the
payload to be delivered to the target site, where for a given
application there is an upper limit in active material or stimulus
to be delivered. Here, if a given mass of active material is coated
to a single projection, then the total number of projections would
be selected such that the total payload is less than this upper
limit.
[0293] In any event, it will be appreciated from the above that it
is typical to use at least 500 projections, but more typically at
least 750, 1000, 2000, 5000, 7500, 10,000, 100,000 projections, and
even as many as 10,000,000 projections.
[0294] Delivery
[0295] Illustrative stimuli or material that can be delivered with
the device of the present invention include any or more of: small
chemical or biochemical compounds including drugs, metabolites,
amino acids, sugars, lipids, saponins, and hormones; macromolecules
such as complex carbohydrates, phospholipids, peptides,
polypeptides, peptidomimetics, and nucleic acids; or other organic
(carbon containing) or inorganic molecules; and particulate matter
including whole cells, bacteria, viruses, virus-like particles,
cell membranes, dendrimers and liposomes.
[0296] In some embodiments, the stimulus or material is selected
from nucleic acids, illustrative examples of which include DNA,
RNA, sense oligonucleotides, antisense oligonucleotides, ribozymes,
small interfering oligonucleotides (siRNAs), micro RNAs (miRNAs),
repeat associated RNAs (rasiRNA), effector RNAs (eRNAs), and any
other oligonucleotides known in the art, which inhibit
transcription and/or translation of a mutated or other detrimental
protein. In illustrative examples of this type, the nucleic acid is
in the form of an expression vector from which a polynucleotide of
interest is expressible. The polynucleotide of interest may encode
a polypeptide or an effector nucleic acid molecule such as sense or
antisense oligonucleotides, siRNAs, miRNAs and eRNAs.
[0297] In other embodiments, the stimulus or material is selected
from peptides or polypeptides, illustrative examples of which
include insulin, proinsulin, follicle stimulating hormone, insulin
like growth factor-1, insulin like growth factor-2, platelet
derived growth factor, epidermal growth factor, fibroblast growth
factors, nerve growth factor, colony stimulating factors,
transforming growth factors, tumor necrosis factor, calcitonin,
parathyroid hormone, growth hormone, bone morphogenic protein,
erythropoietin, hemopoietic growth factors, luteinizing hormone,
glucagon, glucagonlike peptide-1, anti-angiogenic proteins,
clotting factors, anti-clotting factors, atrial natriuretic factor,
plasminogen activators, bombesin, thrombin, enkephalinase, vascular
endothelial growth factor, interleukins, viral antigens, non-viral
antigens, transport proteins, and antibodies. In still other
embodiments, the stimulus or material is selected from receptor
ligands. Illustrative examples of receptors include Fc receptor,
heparin sulfate receptor, vitronectin receptor, Vcam-1 receptor,
hemaglutinin receptor, Pvr receptor, Icam-1 receptor,
decay-accelerating protein (CD55) receptor, Car
(coxsackievirus-adenovirus) receptor, integrin receptor, sialic
acid receptor, HAVCr-1 receptor, low-density lipoprotein receptor,
BGP (biliary glycoprotien) receptor, aminopeptidease N receptor,
MHC class-1 receptor, laminin receptor, nicotinic acetylcholine
receptor, CD56 receptor, nerve growth factor receptor, CD46
receptor, asialoglycoprotein receptor Gp-2, alpha-dystroglycan
receptor, galactosylceramide receptor, Cxcr4 receptor, Glvrl
receptor, Ram-1 receptor, Cat receptor, Tva receptor, BLVRcp1
receptor, MHC class-2 receptor, toll-like receptors (such as TLR-1
to -6) and complement receptors.
[0298] In specific embodiments, the stimuli or material are
selected from antigens including endogenous antigens produced by a
host that is the subject of the stimulus or material delivery or
exogenous antigens that are foreign to that host. The antigens may
be in the form of soluble peptides or polypeptides or
polynucleotides from which an expression product (e.g., protein or
RNA) is producible. Suitable endogenous antigens include, but are
not restricted to, cancer or tumor antigens. Non-limiting examples
of cancer or tumor antigens include antigens from a cancer or tumor
selected from ABL1 proto-oncogene, AIDS related cancers, acoustic
neuroma, acute lymphocytic leukemia, acute myeloid leukemia,
adenocystic carcinoma, adrenocortical cancer, agnogenic myeloid
metaplasia, alopecia, alveolar soft-part sarcoma, anal cancer,
angiosarcoma, aplastic anemia, astrocytoma, ataxia-telangiectasia,
basal cell carcinoma (skin), bladder cancer, bone cancers, bowel
cancer, brain stem glioma, brain and CNS tumors, breast cancer, CNS
tumors, carcinoid tumors, cervical cancer, childhood brain tumors,
childhood cancer, childhood leukemia, childhood soft tissue
sarcoma, chondrosarcoma, choriocarcinoma, chronic lymphocytic
leukemia, chronic myeloid leukemia, colorectal cancers, cutaneous
T-cell lymphoma, dermatofibrosarcoma protuberans, desmoplastic
small round cell tumor, ductal carcinoma, endocrine cancers,
endometrial cancer, ependymoma, oesophageal cancer, Ewing's
Sarcoma, Extra-Hepatic Bile Duct Cancer, Eye Cancer, Eye: Melanoma,
Retinoblastoma, Fallopian Tube cancer, Fanconi anemia,
fibrosarcoma, gall bladder cancer, gastric cancer, gastrointestinal
cancers, gastrointestinal-carcinoid-tumor, genitourinary cancers,
germ cell tumors, gestational-trophoblastic-disease, glioma,
gynecological cancers, haematological malignancies, hairy cell
leukemia, head and neck cancer, hepatocellular cancer, hereditary
breast cancer, histiocytosis, Hodgkin's disease, human
papillomavirus, hydatidiform mole, hypercalcemia, hypopharynx
cancer, intraocular melanoma, islet cell cancer, Kaposi's sarcoma,
kidney cancer, Langerhan's cell histiocytosis, laryngeal cancer,
leiomyosarcoma, leukemia, Li-Fraumeni syndrome, lip cancer,
liposarcoma, liver cancer, lung cancer, lymphedema, lymphoma,
Hodgkin's lymphoma, non-Hodgkin's lymphoma, male breast cancer,
malignant-rhabdoid tumor of kidney, medulloblastoma, melanoma,
Merkel cell cancer, mesothelioma, metastatic cancer, mouth cancer,
multiple endocrine neoplasia, mycosis fungoides, myelodysplastic
syndromes, myeloma, myeloproliferative disorders, nasal cancer,
nasopharyngeal cancer, nephroblastoma, neuroblastoma,
neurofibromatosis, Nijmegen breakage syndrome, non-melanoma skin
cancer, non-small-cell-lung-cancer (NSCLC), ocular cancers,
esophageal cancer, oral cavity cancer, oropharynx cancer,
osteosarcoma, ostomy ovarian cancer, pancreas cancer, paranasal
cancer, parathyroid cancer, parotid gland cancer, penile cancer,
peripheral-neuroectodermal tumors, pituitary cancer, polycythemia
vera, prostate cancer, rare cancers and associated disorders, renal
cell carcinoma, retinoblastoma, rhabdomyosarcoma, Rothmund-Thomson
syndrome, salivary gland cancer, sarcoma, schwannoma, Sezary
syndrome, skin cancer, small cell lung cancer (SCLC), small
intestine cancer, soft tissue sarcoma, spinal cord tumors,
squamous-cell-carcinoma-(skin), stomach cancer, synovial sarcoma,
testicular cancer, thymus cancer, thyroid cancer,
transitional-cell-cancer-(bladder),
transitional-cell-cancer-(renal-pelvis-/-ureter), trophoblastic
cancer, urethral cancer, urinary system cancer, uroplakins, uterine
sarcoma, uterus cancer, vaginal cancer, vulva cancer, Waldenstrom's
macroglobulinemia, Wilms' tumor. In certain embodiments, the cancer
or tumor relates to melanoma. Illustrative examples of
melanoma-related antigens include melanocyte differentiation
antigen (e.g., gp100, MART, Melan-A/MART-1, TRP-1, Tyros, TRP2,
MC1R, MUC1F, MUC1R or a combination thereof) and melanoma-specific
antigens (e.g., BAGE, GAGE-1, gp100In4, MAGE-1 (e.g., GenBank
Accession No. X54156 and AA494311), MAGE-3, MAGE4, PRAME, TRP2IN2,
NYNSO1a, NYNSO1b, LAGE1, p97 melanoma antigen (e.g., GenBank
Accession No. M12154) p5 protein, gp75, oncofetal antigen, GM2 and
GD2 gangliosides, cdc27, p21ras, gp100.sup.Pmel117 or a combination
thereof. Other tumor-specific antigens include, but are not limited
to: etv6, aml1, cyclophilin b (acute lymphoblastic leukemia);
Ig-idiotype (B cell lymphoma); E-cadherin, .alpha.-catenin,
.beta.-catenin, .gamma.-catenin, p120ctn (glioma); p21ras (bladder
cancer); p21ras (biliary cancer); MUC family, HER2/neu, c-erbB-2
(breast cancer); p53, p21ras (cervical carcinoma); p21ras,
HER2/neu, c-erbB-2, MUC family, Cripto-1protein, Pim-1 protein
(colon carcinoma); Colorectal associated antigen
(CRC)-CO17-1A/GA733, APC (colorectal cancer); carcinoembryonic
antigen (CEA) (colorectal cancer; choriocarcinoma); cyclophilin b
(epithelial cell cancer); HER2/neu, c-erbB-2, ga733 glycoprotein
(gastric cancer); .alpha.-fetoprotein (hepatocellular cancer);
Imp-1, EBNA-1 (Hodgkin's lymphoma); CEA, MAGE-3, NY-ESO-1 (lung
cancer); cyclophilin b (lymphoid cell-derived leukemia); MUC
family, p21ras (myeloma); HER2/neu, c-erbB-2 (non-small cell lung
carcinoma); Imp-1, EBNA-1 (nasopharyngeal cancer); MUC family,
HER2/neu, c-erbB-2, MAGE-A4, NY-ESO-1 (ovarian cancer); Prostate
Specific Antigen (PSA) and its antigenic epitopes PSA-1, PSA-2, and
PSA-3, PSMA, HER2/neu, c-erbB-2, ga733 glycoprotein (prostate
cancer); HER2/neu, c-erbB-2 (renal cancer); viral products such as
human papillomavirus proteins (squamous cell cancers of the cervix
and esophagus); NY-ESO-1 (testicular cancer); and HTLV-1 epitopes
(T cell leukemia).
[0299] Foreign antigens are suitably selected from transplantation
antigens, allergens as well as antigens from pathogenic organisms.
Transplantation antigens can be derived from donor cells or tissues
from, e.g., heart, lung, liver, pancreas, kidney, neural graft
components, or from the donor antigen-presenting cells bearing MHC
loaded with self antigen in the absence of exogenous antigen.
[0300] Non-limiting examples of allergens include Fel d 1 (i.e.,
the feline skin and salivary gland allergen of the domestic cat
Felis domesticus, the amino acid sequence of which is disclosed
International Publication WO 91/06571), Der p I, Der p II, Der fI
or Der fII (i.e., the major protein allergens from the house dust
mite dermatophagoides, the amino acid sequence of which is
disclosed in International Publication WO 94/24281). Other
allergens may be derived, for example from the following: grass,
tree and weed (including ragweed) pollens; fungi and molds; foods
such as fish, shellfish, crab, lobster, peanuts, nuts, wheat
gluten, eggs and milk; stinging insects such as bee, wasp, and
hornet and the chirnomidae (non-biting midges); other insects such
as the housefly, fruitfly, sheep blow fly, screw worm fly, grain
weevil, silkworm, honeybee, non-biting midge larvae, bee moth
larvae, mealworm, cockroach and larvae of Tenibrio molitor beetle;
spiders and mites, including the house dust mite; allergens found
in the dander, urine, saliva, blood or other bodily fluid of
mammals such as cat, dog, cow, pig, sheep, horse, rabbit, rat,
guinea pig, mouse and gerbil; airborne particulates in general;
latex; and protein detergent additives.
[0301] Exemplary pathogenic organisms include, but are not limited
to, viruses, bacteria, fungi parasites, algae and protozoa and
amoebae. Illustrative viruses include viruses responsible for
diseases including, but not limited to, measles, mumps, rubella,
poliomyelitis, hepatitis A, B (e.g., GenBank Accession No. E02707),
and C (e.g., GenBank Accession No. E06890), as well as other
hepatitis viruses, influenza, adenovirus (e.g., types 4 and 7),
rabies (e.g., GenBank Accession No. M34678), yellow fever,
Epstein-Barr virus and other herpesviruses such as papillomavirus,
Ebola virus, influenza virus, Japanese encephalitis (e.g., GenBank
Accession No. E07883), dengue (e.g., GenBank Accession No. M24444),
hantavirus, Sendai virus, respiratory syncytial virus,
othromyxoviruses, vesicular stomatitis virus, visna virus,
cytomegalovirus and human immunodeficiency virus (HIV) (e.g.,
GenBank Accession No. U18552). Any suitable antigen derived from
such viruses are useful in the practice of the present invention.
For example, illustrative retroviral antigens derived from HIV
include, but are not limited to, antigens such as gene products of
the gag, pol, and env genes, the Nef protein, reverse
transcriptase, and other HIV components. Illustrative examples of
hepatitis viral antigens include, but are not limited to, antigens
such as the S, M, and L proteins of hepatitis B virus, the pre-S
antigen of hepatitis B virus, and other hepatitis, e.g., hepatitis
A, B, and C, viral components such as hepatitis C viral RNA.
Illustrative examples of influenza viral antigens include, but are
not limited to, antigens such as hemagglutinin and neuraminidase
and other influenza viral components. Illustrative examples of
measles viral antigens include, but are not limited to, antigens
such as the measles virus fusion protein and other measles virus
components. Illustrative examples of rubella viral antigens
include, but are not limited to, antigens such as proteins E1 and
E2 and other rubella virus components; rotaviral antigens such as
VP7sc and other rotaviral components. Illustrative examples of
cytomegaloviral antigens include, but are not limited to, antigens
such as envelope glycoprotein B and other cytomegaloviral antigen
components. Non-limiting examples of respiratory syncytial viral
antigens include antigens such as the RSV fusion protein, the M2
protein and other respiratory syncytial viral antigen components.
Illustrative examples of herpes simplex viral antigens include, but
are not limited to, antigens such as immediate early proteins,
glycoprotein D, and other herpes simplex viral antigen components.
Non-limiting examples of varicella zoster viral antigens include
antigens such as 9P1, gpII, and other varicella zoster viral
antigen components. Non-limiting examples of Japanese encephalitis
viral antigens include antigens such as proteins E, M-E, M-E-NS 1,
NS 1, NS 1-NS2A, 80%E, and other Japanese encephalitis viral
antigen components. Representative examples of rabies viral
antigens include, but are not limited to, antigens such as rabies
glycoprotein, rabies nucleoprotein and other rabies viral antigen
components. Illustrative examples of papillomavirus antigens
include, but are not limited to, the L1 and L2 capsid proteins as
well as the E6/E7 antigens associated with cervical cancers, See
Fundamental Virology, Second Edition, eds. Fields, B. N. and Knipe,
D. M., 1991, Raven Press, New York, for additional examples of
viral antigens.
[0302] Illustrative examples of fungi include Acremonium spp.,
Aspergillus spp., Basidiobolus spp., Bipolaris spp., Blastomyces
dermatidis, Candida spp., Cladophialophora carrionii, Coccoidiodes
immitis, Conidiobolus spp., Cryptococcus spp., Curvularia spp.,
Epidermophyton spp., Exophiala jeanselmei, Exserohilum spp.,
Fonsecaea compacta, Fonsecaea pedrosoi, Fusarium oxysporum,
Fusarium solani, Geotrichum candidum, Histoplasma capsulatum var.
capsulatum, Histoplasma capsulatum var. duboisii, Hortaea
werneckii, Lacazia loboi, Lasiodiplodia theobromas, Leptosphaeria
senegalensis, Madurella grisea, Madurella mycetomatis, Malassezia
furfur, Microsporum spp., Neotestudina rosatii, Onychocola
canadensis, Paracoccidioides brasiliensis, Phialophora verrucosa,
Piedraia hortae, Piedra iahortae, Pityriasis versicolor,
Pseudallesheria boydii, Pyrenochaeta romeroi, Rhizopus arrhizus,
Scopulariopsis brevicaulis, Scytalidium dimidiatum, Sporothrix
schenckii, Trichophyton spp., Trichosporon spp., Zygomcete fungi,
Absidia corymbifera, Rhizomucor pusillus and Rhizopus arrhizus.
Thus, representative fungal antigens that can be used in the
compositions and methods of the present invention include, but are
not limited to, candida fungal antigen components; histoplasma
fungal antigens such as heat shock protein 60 (HSP60) and other
histoplasma fungal antigen components; cryptococcal fungal antigens
such as capsular polysaccharides and other cryptococcal fungal
antigen components; coccidiodes fungal antigens such as spherule
antigens and other coccidiodes fungal antigen components; and tinea
fungal antigens such as trichophytin and other coccidiodes fungal
antigen components.
[0303] Illustrative examples of bacteria include bacteria that are
responsible for diseases including, but not restricted to,
diphtheria (e.g., Corynebacterium diphtheria), pertussis (e.g.,
Bordetella pertussis, GenBank Accession No. M35274), tetanus (e.g.,
Clostridium tetani, GenBank Accession No. M64353), tuberculosis
(e.g., Mycobacterium tuberculosis), bacterial pneumonias (e.g.,
Haemophilus influenzae.), cholera (e.g., Vibrio cholerae), anthrax
(e.g., Bacillus anthracis), typhoid, plague, shigellosis (e.g.,
Shigella dysenteriae), botulism (e.g., Clostridium botulinum),
salmonellosis (e.g., GenBank Accession No. L03833), peptic ulcers
(e.g., Helicobacter pylori), Legionnaire's Disease, Lyme disease
(e.g., GenBank Accession No. U59487). Other pathogenic bacteria
include Escherichia coli, Clostridium perfringens, Pseudomonas
aeruginosa, Staphylococcus aureus and Streptococcus pyogenes. Thus,
bacterial antigens which can be used in the compositions and
methods of the invention include, but are not limited to: pertussis
bacterial antigens such as pertussis toxin, filamentous
hemagglutinin, pertactin, F M2, FIM3, adenylate cyclase and other
pertussis bacterial antigen components; diphtheria bacterial
antigens such as diphtheria toxin or toxoid and other diphtheria
bacterial antigen components; tetanus bacterial antigens such as
tetanus toxin or toxoid and other tetanus bacterial antigen
components, streptococcal bacterial antigens such as M proteins and
other streptococcal bacterial antigen components; gram-negative
bacilli bacterial antigens such as lipopolysaccharides and other
gram-negative bacterial antigen components; Mycobacterium
tuberculosis bacterial antigens such as mycolic acid, heat shock
protein 65 (HSP65), the 30 kDa major secreted protein, antigen 85A
and other mycobacterial antigen components; Helicobacter pylori
bacterial antigen components, pneumococcal bacterial antigens such
as pneumolysin, pneumococcal capsular polysaccharides and other
pnermiococcal bacterial antigen components; Haemophilus influenza
bacterial antigens such as capsular polysaccharides and other
Haemophilus influenza bacterial antigen components; anthrax
bacterial antigens such as anthrax protective antigen and other
anthrax bacterial antigen components; rickettsiae bacterial
antigens such as rompA and other rickettsiae bacterial antigen
component. Also included with the bacterial antigens described
herein are any other bacterial, mycobacterial, mycoplasmal,
rickettsial, or chlamydial antigens.
[0304] Illustrative examples of protozoa include protozoa that are
responsible for diseases including, but not limited to, malaria
(e.g., GenBank Accession No. X53832), hookworm, onchocerciasis
(e.g., GenBank Accession No. M27807), schistosomiasis (e.g.,
GenBank Accession No. LOS 198), toxoplasmosis, trypanosomiasis,
leishmaniasis, giardiasis (GenBank Accession No. M33641),
amoebiasis, filariasis (e.g., GenBank Accession No. J03266),
borreliosis, and trichinosis. Thus, protozoal antigens which can be
used in the compositions and methods of the invention include, but
are not limited to: plasmodium falciparum antigens such as
merozoite surface antigens, sporozoite surface antigens,
circumsporozoite antigens, gametocyte/gamete surface antigens,
blood-stage antigen pf 155/RESA and other plasmodial antigen
components; toxoplasma antigens such as SAG-1, p30 and other
toxoplasmal antigen components; schistosomae antigens such as
glutathione-S-transferase, paramyosin, and other schistosomal
antigen components; leishmania major and other leishmaniae antigens
such as gp63, lipophosphoglycan and its associated protein and
other leishmanial antigen components; and trypanosoma cruzi
antigens such as the 75-77 kDa antigen, the 56 kDa antigen and
other trypanosomal antigen components.
[0305] The present invention also contemplates toxin components as
antigens. Illustrative examples of toxins include, but are not
restricted to, staphylococcal enterotoxins, toxic shock syndrome
toxin; retroviral antigens (e.g., antigens derived from HIV),
streptococcal antigens, staphylococcal enterotoxin-A (SEA),
staphylococcal enterotoxin-B (SEB), staphylococcal
enterotoxin.sub.1-3 (SE.sub.1-3), staphylococcal enterotoxin-D
(SED), staphylococcal enterotoxin-E (SEE) as well as toxins derived
from mycoplasma, mycobacterium, and herpes viruses.
[0306] In specific embodiments, the antigen is delivered to
antigen-presenting cells. Such antigen-presenting cells include
professional or facultative antigen-presenting cells. Professional
antigen-presenting cells function physiologically to present
antigen in a form that is recognized by specific T cell receptors
so as to stimulate or energies a T lymphocyte or B lymphocyte
mediated immune response. Professional antigen-presenting cells not
only process and present antigens in the context of the major
histocompatability complex (MHC), but also possess the additional
immunoregulatory molecules required to complete T cell activation
or induce a tolerogenic response. Professional antigen-presenting
cells include, but are not limited to, macrophages, monocytes, B
lymphocytes, cells of myeloid lineage, including
monocytic-granulocytic-DC precursors, marginal zone Kupffer cells,
microglia, T cells, Langerhans cells and dendritic cells including
interdigitating dendritic cells and follicular dendritic cells.
Non-professional or facultative antigen-presenting cells typically
lack one or more of the immunoregulatory molecules required to
complete T lymphocyte activation or energy. Examples of
non-professional or facultative antigen-presenting cells include,
but are not limited to, activated T lymphocytes, eosinophils,
keratinocytes, astrocytes, follicular cells, microglial cells,
thymic cortical cells, endothelial cells, Schwann cells, retinal
pigment epithelial cells, myoblasts, vascular smooth muscle cells,
chondrocytes, enterocytes, thymocytes, kidney tubule cells and
fibroblasts. In some embodiments, the antigen-presenting cell is
selected from monocytes, macrophages, B lymphocytes, cells of
myeloid lineage, dendritic cells or Langerhans cells. In certain
advantageous embodiments, the antigen-presenting cell expresses
CD11c and includes a dendritic cell or Langerhans cell. In some
embodiments the antigen-presenting cell stimulates an immune
response. In other embodiments, the antigen-presenting cell induces
a tolerogenic response.
[0307] The delivery of exogenous antigen to an antigen-presenting
cell can be enhanced by methods known to practitioners in the art.
For example, several different strategies have been developed for
delivery of exogenous antigen to the endogenous processing pathway
of antigen-presenting cells, especially dendritic cells. These
methods include insertion of antigen into pH-sensitive liposomes
(Zhou and Huang, 1994, Immunomethods, 4:229-235), osmotic lysis of
pinosomes after pinocytic uptake of soluble antigen (Moore et al.,
1988, Cell, 54:777-785), coupling of antigens to potent adjuvants
(Aichele et al., 1990, J. Exp. Med., 171: 1815-1820; Gao et al.,
1991, J. Immunol., 147: 3268-3273; Schulz et al., 1991, Proc. Natl.
Acad. Sci. USA, 88: 991-993; Kuzu et al., 1993, Euro. J. Immunol.,
23: 1397-1400; and Jondal et al., 1996, Immunity 5: 295-302) and
apoptotic cell delivery of antigen (Albert et al. 1998, Nature
392:86-89; Albert et al. 1998, Nature Med. 4:1321-1324; and in
International Publications WO 99/42564 and WO 01/85207).
Recombinant bacteria (e.g., E. coli) or transfected host mammalian
cells may be pulsed onto dendritic cells (as particulate antigen,
or apoptotic bodies respectively) for antigen delivery. Recombinant
chimeric virus-like particles (VLPs) have also been used as
vehicles for delivery of exogenous heterologous antigen to the MHC
class I processing pathway of a dendritic cell line (Bachmann et
al., 1996, Eur. J. Immunol., 26(11): 2595-2600).
[0308] Alternatively, or in addition, an antigen may be linked to,
or otherwise associated with, a cytolysin to enhance the transfer
of the antigen into the cytosol of an antigen-presenting cell of
the invention for delivery to the MHC class I pathway. Exemplary
cytolysins include saponin compounds such as saponin-containing
Immune Stimulating Complexes (ISCOMs) (see, e.g., Cox and Coulter,
1997, Vaccine 15(3): 248-256 and U.S. Pat. No. 6,352,697),
phospholipases (see, e.g., Camilli et al., 1991, J. Exp. Med. 173:
751-754), pore-forming toxins (e.g., an a-toxin), natural
cytolysins of gram-positive bacteria, such as listeriolysin O (LLO,
e.g., Mengaud et al., 1988, Infect. Immun. 56: 766-772 and Portnoy
et al., 1992, Infect. Immun. 60: 2710-2717), streptolysin O (SLO,
e.g., Palmer et al., 1998, Biochemistry 37(8): 2378-2383) and
perfringolysin O (PFO, e.g., Rossjohn et al., Cell 89(5): 685-692).
Where the antigen-presenting cell is phagosomal, acid activated
cytolysins may be advantageously used. For example, listeriolysin
exhibits greater pore-forming ability at mildly acidic pH (the pH
conditions within the phagosome), thereby facilitating delivery of
vacuole (including phagosome and endosome) contents to the
cytoplasm (see, e.g., Portnoy et al., Infect. Immun. 1992, 60:
2710-2717).
[0309] The cytolysin may be provided together with a pre-selected
antigen in the form of a single composition or may be provided as a
separate composition, for contacting the antigen-presenting cells.
In one embodiment, the cytolysin is fused or otherwise linked to
the antigen, wherein the fusion or linkage permits the delivery of
the antigen to the cytosol of the target cell. In another
embodiment, the cytolysin and antigen are provided in the form of a
delivery vehicle such as, but not limited to, a liposome or a
microbial delivery vehicle selected from virus, bacterium, or
yeast. Suitably, when the delivery vehicle is a microbial delivery
vehicle, the delivery vehicle is non-virulent. In a preferred
embodiment of this type, the delivery vehicle is a non-virulent
bacterium, as for example described by Portnoy et al. in U.S. Pat.
No. 6,287,556, comprising a first polynucleotide encoding a
non-secreted functional cytolysin operably linked to a regulatory
polynucleotide which expresses the cytolysin in the bacterium, and
a second polynucleotide encoding one or more pre-selected antigens.
Non-secreted cytolysins may be provided by various mechanisms,
e.g., absence of a functional signal sequence, a secretion
incompetent microbe, such as microbes having genetic lesions (e.g.,
a functional signal sequence mutation), or poisoned microbes, etc.
A wide variety of nonvirulent, non-pathogenic bacteria may be used;
preferred microbes are relatively well characterized strains,
particularly laboratory strains of E. coli, such as MC4100, MC1061,
DH5.alpha., etc. Other bacteria that can be engineered for the
invention include well-characterized, nonvirulent, non-pathogenic
strains of Listeria monocytogenes, Shigella flexneri,
mycobacterium, Salmonella, Bacillus subtilis, etc. In a particular
embodiment, the bacteria are attenuated to be non-replicative,
non-integrative into the host cell genome, and/or non-motile inter-
or intra-cellularly.
[0310] The delivery vehicles described above can be used to deliver
one or more antigens to virtually any antigen-presenting cell
capable of endocytosis of the subject vehicle, including phagocytic
and non-phagocytic antigen-presenting cells. In embodiments when
the delivery vehicle is a microbe, the subject methods generally
require microbial uptake by the target cell and subsequent lysis
within the antigen-presenting cell vacuole (including phagosomes
and endosomes).
[0311] In other embodiments, the antigen is produced inside the
antigen-presenting cell by introduction of a suitable expression
vector as for example described above. The antigen-encoding portion
of the expression vector may comprise a naturally-occurring
sequence or a variant thereof, which has been engineered using
recombinant techniques. In one example of a variant, the codon
composition of an antigen-encoding polynucleotide is modified to
permit enhanced expression of the antigen in a target cell or
tissue of choice using methods as set forth in detail in
International Publications WO 99/02694 and WO 00/42215. Briefly,
these methods are based on the observation that translational
efficiencies of different codons vary between different cells or
tissues and that these differences can be exploited, together with
codon composition of a gene, to regulate expression of a protein in
a particular cell or tissue type. Thus, for the construction of
codon-optimized polynucleotides, at least one existing codon of a
parent polynucleotide is replaced with a synonymous codon that has
a higher translational efficiency in a target cell or tissue than
the existing codon it replaces. Although it is preferable to
replace all the existing codons of a parent nucleic acid molecule
with synonymous codons which have that higher translational
efficiency, this is not necessary because increased expression can
be accomplished even with partial replacement. Suitably, the
replacement step affects 5, 10, 15, 20, 25, 30%, more preferably
35, 40, 50, 60, 70% or more of the existing codons of a parent
polynucleotide.
[0312] The expression vector for introduction into the
antigen-presenting cell will be compatible therewith such that the
antigen-encoding polynucleotide is expressible by the cell. For
example, expression vectors of this type can be derived from viral
DNA sequences including, but not limited to, adenovirus,
adeno-associated viruses, herpes-simplex viruses and retroviruses
such as B, C, and D retroviruses as well as spumaviruses and
modified lentiviruses. Suitable expression vectors for transfection
of animal cells are described, for example, by Wu and Ataai (2000,
Curr. Opin. Biotechnol. 11(2):205-208), Vigna and Naldini (2000, J.
Gene Med. 2(5):308-316), Kay, et al. (2001, Nat. Med. 7(1):33-40),
Athanasopoulos, et al. (2000, Int. J. Mol. Med. 6(4):363-375) and
Walther and Stein (2000, Drugs 60(2):249-271).
[0313] Experimental Results
[0314] Preliminary data in a mouse model, using a virus as a
delivery vehicle to the skin using an embodiment of the patches 100
described above elicited significantly higher cytotoxic
T-lymphocyte responses than conventional intradermal injection of
the same antigenic preparation, with the same payload.
[0315] Accordingly, the above described examples provide a device
for the delivery of a bioactive material (agent) or other stimulus
to an internal site in the body, comprising a plurality (number) of
projections that can penetrate a body surface so as to deliver the
bioactive material or stimulus to the required site.
[0316] In one example, the number of projections is selected to be
at least 500, to thereby induce a biological response. Typically
the exact number of projections is determined in accordance with
the above described equations, thereby maximizing the chance of
delivery of material or stimulus to the target.
[0317] The delivery end portion of the projection may also be
dimensioned so as to be capable of insertion into targeted cells to
deliver the bioactive material or stimulus without appreciable
damage to the targeted cells or specific sites therein. Thus, the
dimensions of the delivery end portion of the microprojections,
including the length or diameter, can be selected which enables
delivery of the agent or stimulus to targeted cells and internal
components within cells.
[0318] The nanoneedles are typically solid (non-hollow) in
cross-section. This leads to a number of technical advantages which
include: reliable delivery of bioactive material or stimulus; ease
and cost of manufacturing, and increased strength.
Variations
[0319] A number of variations and options for use with the above
described devices will now be described.
[0320] Herein, the terms "projection", "micro-nanoprojection",
"nanoneedle", "nanoprojection", "needle", "rod", etc., are used
interchangeably to describe the solid projections.
[0321] In cases where a material or agent is to be transported,
projections may be coated on the outside of the nanoneedles. This
provides a higher probability of delivering the coating to the
depth of interest compared to microparticle delivery from a gene
gun and thus is more efficient.
[0322] A further feature is that the nanoneedles may be used for
delivery not only through the skin but through other body surfaces,
including mucosal surfaces, to cellular sites below the outer layer
or layers of such surfaces. The term "internal site", as used
herein, is to be understood as indicating a site below the outer
layer(s) of skin and other tissues for which the devices of the
present invention are to be used.
[0323] Furthermore, these nanoneedles may be used to deliver
stimuli to cells or cell components other than those resulting from
the administration of bioactive agents such as drugs and antigenic
materials for example. Mere penetration of cellular sites with
nanoneedles may be sufficient to induce a beneficial response, as
indicated hereinafter.
[0324] The device is suitable for intracellular delivery. The
device is suitable for delivery to specific organelles within
cells. Examples of organelles to which the device can be applied
include a cell nucleus, endoplasmic reticulum, ribosome, or
lysosome for example.
[0325] In one embodiment the device is provided having a needle
support section, that is to say the projections comprise a suitable
support section, of sufficient length to reach the desired site and
a (needle) delivery end section having a length no greater than 20
microns and a maximum width no greater than 5 microns, preferably
no greater than 2 microns.
[0326] In one example, the maximum width of the delivery end
section is no greater than 1000 nm, even more preferably the
maximum width of the delivery end section is no greater than 500
nm.
[0327] In one embodiment, the device can be used for delivery
intradermally. This device may have a needle support section, that
is to say the projections comprise a suitable support section, of
length at least 10 microns and a (needle) delivery end section
having a length no greater than 20 microns and a maximum width no
greater than 5 microns, preferably no greater than 2 microns.
[0328] The maximum width of the delivery end section is usually no
greater than 1000 nm, even more preferably the maximum width of the
delivery end section is no greater than 500 nm.
[0329] In a further embodiment, the device is for mucosal delivery.
This device may have a needle support section, that is to say the
projections comprise a suitable support section, of sufficient
length to reach the desired site, such as of length at least 100
microns and a (needle) delivery end section having a length no
greater than 20 microns and a maximum width no greater than 5
microns, preferably no greater than 2 microns.
[0330] In one embodiment, the device of the invention is for
delivery to lung or other internal organ or tissue. In a further
embodiment, the device is for in-vitro delivery to tissue, cell
cultures, cell lines, organs, artificial tissues and tissue
engineered products. This device typically has a needle support
section, that is to say the projections comprise a suitable support
section, of length at least 5 microns and a needle delivery end
section having a length no greater than 20 microns and a maximum
width no greater than 5 microns, preferably no greater than 2
microns.
[0331] In one embodiment, the device comprises projections in which
the (needle) delivery end section and support length, that is to
say the "needle support section", is coated with a bioactive
material across the whole or part of its length. The (needle)
delivery end section and support length may be coated on selective
areas thereof. This may depend upon the bioactive material being
used or the target selected for example.
[0332] In a further embodiment, a bioactive material is releasably
incorporated into the material of which the needle, or projection,
is composed. All, or part of the projection may be constructed of a
biocompatible, biodegradable polymer (such as Poly Lactic Acid
(PLA), PolyGlycolic Acid (PGA) or PGLA or Poly Glucleic Acid),
which is formulated with the bioactive material of choice. The
projections may then be inserted into the appropriate target site
and, as they dissolve, the bioactive material will enter the
organelle(s)/cells.
[0333] In one example, at least the delivery end section of the
needle is composed of a biodegradable material.
[0334] In an alternative embodiment of the invention, a device is
also provided in which the needle has no bioactive material on or
within it. The targeted cell or organelle is perturbed/stimulated
by the physical disruption caused by the (delivery end of the)
nanoneedle (projection). This physical stimulus may, for example,
be coupled with electric stimulus as a form of specific
nanoelectroporation of particular organelles or the cell.
[0335] The bioactive material or stimulus delivered by the device
of the invention may be any suitable material or stimulus which
gives the desired effect at the target site.
[0336] Examples of bioactive materials, which are not intended to
be limiting with respect to the invention include polynucleotides
and nucleic acid or protein molecules, antigens, allergens,
adjuvants, molecules, elements or compounds. In addition, the
device may be coated with materials such as biosensors, nanosensors
or MEMS.
[0337] In one aspect, the device is provided in the form of a patch
containing a plurality of needles (projections) for application to
a body surface. A multiplicity of projections can allow multiple
cells and organelles to be targeted and provided with a bioactive
material or stimulus at the same time. The patch may be of any
suitable shape, such as square or round for example. The overall
number of projections per patch depends upon the particular
application in which the device is to be used. Preferably, the
patch has at least 10 needles per mm, and more preferably at least
100 needles per mm.sup.2. Considerations and specific examples of
such a patch are provided in more detail below.
[0338] Examples of specific manufacturing steps used to fabricate
the device are described in greater detail below. In one preferred
aspect, the device of the invention is constructed from
biocompatible materials such as Titanium, Gold, Silver or Silicon,
for example. This may be the entire device, or alternatively it may
only be the projections or the delivery end section of the
projections which are made from the biocompatible materials.
[0339] One manufacturing method for the device utilizes the Deep
Reactive Ion Etching (DRIE) of the patterns direct from silicon
wafers, see the construction section below.
[0340] Another manufacturing method for the device utilizes
manufacturing from a male template constructed with X-ray
lithography, electrodeposition and molding (LIGA). The templates
are then multiply inserted into a soft polymer to produce a
plurality of masks. The masks are then vacuum deposited/sputtered
with the material of choice for the nanoprojections, such as
titanium, gold, silver, or tungsten. Magnetron sputtering may also
be applied, see the construction section below.
[0341] An alternative means for producing masks is with 2 photon
Stereolithography, a technique which is known in the art and is
described in more detail below.
[0342] In one embodiment, the device is constructed of silicon.
[0343] The device may be for a single use or may be used and then
recoated with the same or a different bioactive material or other
stimulus, for example.
[0344] In one embodiment, the device comprises projections which
are of differing lengths and/or diameters (or thicknesses depending
on the shape of the projections) to allow targeting of different
targets within the same use of the device.
[0345] An example of the practical application of such a device is
explained in further detail below (see section 6.2.4).
[0346] Also provided throughout the specification are numerous uses
of the device, which has many useful medical applications in the
treatment of a number of diseases.
Further Examples
[0347] Further examples will now be described in more detail. For
the purpose of these examples, the explanation will focus on the
targeting of cells, cell organelles or cell nuclei. However it will
be appreciated that the techniques may be used to deliver
material/stimulus to any suitable specific target.
1. Specific Targets for Delivery
[0348] In one example, the target of interest is within the cells.
The position and shape of key organelles within the cells are shown
in FIG. 15. All eucaryotic cells have the same basic set of
membrane enclosed organelles. The number and volume of the key
organelles varies with cell type. As to the targeting of specific
organelles, there is a probability attached to each event on the
basis of volume. Consider the non-specific targeting of the cell
(i.e., at the correct depth, but without the precise targeting with
the aid of imaging techniques). The probability of targeting the
nucleus, for example, in a cell is given by the volume of the
nucleus vs. the remainder of the cell.
[0349] In Table 1 below, the scale of organelles and their mass
fraction and number are listed. The primary data for this summary
is from "Genes VI" by Benjamin Lewin and "The Molecular Biology of
the Cell" Alberts et al., 4th Ed. This information pertains to the
Liver Cell. Also listed in Table 1 are example applications and
that may be induced or enhanced as a result of targeting these
organelles.
[0350] Working in increasing scale, the starting point is the cell
membrane, which is .about.10 nm thick. In piercing these membranes
with a minimal disruption to the viability of the cell, a range of
drugs, vaccines and other compounds can be delivered to cells.
Specifically, the Endoplasmic Reticulum (a convoluted envelope, 75
nm in thickness) may be targeted within RNA to transfect cells in
the areas of Vaccination or Gene Therapy. Lysosomes, which are
200-500 nm in size can be targeted for release of enzymes to induce
autolysis (cell death). For an effective cellular response (MHC
Class I) with DNA vaccination and gene therapy, it is important
that the DNA material is delivered intact to the cell nucleus.
[0351] Most of these organelles are of the submicron scale.
TABLE-US-00002 TABLE 1 The scale, number and potential targets of
key organelles within cells.
(http://niko.unl.edu/bs101/notes/sizes.html and "Molecular Biology
of the Cell" 4''' Ed, Alberts et al.) Mass/ Volume Number per
Utility as Scale Organelle Fraction cell target Application ~0.1 nm
Hydrogen Atom ~0.8 nm Amino Acid ~2 nm DNA Alpha ~2% (diameter)
helix ~2 nm mRNA ~2% ~2,500 ~2 nm tRNA ~3% ~160,000 ~2 nm RNA ~21%
~4 nm Globular Protein ~10 nm Cell ~10% Use To enhance membranes
nanoneedles drug/vaccine to pierce delivery of these cells through
membranes perfusion with minimal disruption to the cell. ~11 nm
Ribosome ~9% ~25 nm Microtubule diameter ~75 nm Endoplasmic ~15%
Target with Vaccination, (envelope reticulum RNA, gene therapy
thickness) (smooth, mRNA, for rough, iRNA for up cancerous Golgi)
regulating or cells. interfering the production of proteins ~100 nm
Large Virus ~200-500 Lysosomes ~1% Pierce the Inducing cell nm
lysosomes to death in release cancerous enzymes and cells. induces
autolysis (self digestion of the cell). ~3 .mu.m .times.
Mitochondri ~22% 200 nm on ~3-6 .mu.m Nucleus ~6% (Liver) 1 Target
for DNA 16% (LC) DNA vaccination delivery to or gene the cell
therapy. directly and/or disturb the double membrane. ~10-30 .mu.m
Cell Langerhans (diameter) cells are ~10 in diameter.
2. Routes of Delivery to Cells
[0352] The type and scale of organelles within cells has been
presented. Now the location of these cells within the tissue is
identified. The selected tissue routes are.
[0353] Intradermal
[0354] Mucosal
[0355] Lung
[0356] Internal tissues
[0357] In-vitro sites.
2.1 Intradermal Delivery
[0358] The skin is one convenient route for drug and vaccine
delivery. A schematic of the skin is shown in FIG. 16. A full
description of the anatomy of the skin is given in textbooks. A
closer view of a histology section of skin is shown in FIG. 17 in a
photomicrograph. Respective thicknesses of layers vary between
species and sites.
[0359] A key barrier to many drugs and vaccines is the Stratum
Corneum (SC). In humans, this barrier is 10-20 .mu.m thick with
large variations from site-to-site with different ages and sexes.
Below the SC is the Viable Epidermis (VE), which is typically 40-60
.mu.m thick in humans. Within the VE are immunologically sensitive
Langerhans Cells (LC). FIG. 17 shows a stained LC, marked as "L",
residing above the basal layer. The spatial distribution of LC is
illustrated in a 3D distribution of LC in a mouse, FIG. 18 (Kendall
M. A. F., Mulholland W. J., Tirlapur U. K., Arbuthnott E. S., and
Armitage, M. (2003) "Targeted delivery of micro-particles to
epithelial cells for immunotherapy and vaccines: an experimental
and probabilistic study", The 6th International Conference on
Cellular Engineering, Sydney, August 20-22).
[0360] There are typically about 1000 LC per square mm and they
reside just above the basement membrane of the viable epidermis
(i.e., 30-80 .mu.m deep). Importantly, the depth of LC varies
significantly with the biological variability of the tissue,
including rete ridges etc.
[0361] One objective in vaccination and gene therapy applications
is to target LC residing 30-80 .mu.m below the skin surface. In
order to do this, the SC and cell membranes are to be breached.
Furthermore, the organelles within the LC are to be targeted to
elicit particular responses. One example is in the triggering of
MHC I responses, whereby intact DNA is to be delivered to cell the
nucleus. Alternatively, mRNA can be delivered to the Endoplasmic
Reticulum or the cytoplasm.
[0362] Similarly, in the treatment of skin cancers such as Squamous
Cell Carcinoma, cancerous cells within the VE may be directly
targeted.
[0363] In some applications it may be beneficial to target cells
deeper into the dermis. For example, in Basal Cell Carcinoma, the
cells to be targeted are deeper in the tissue, approaching hundreds
of microns (600 82 m to 800 .mu.m) (British Journal of Dermatology
149(5) Page 1035-2003).
2.2 Mucosal Delivery
[0364] FIG. 19A shows a photomicrograph of a histology section of
the mucosa and FIG. 19B shows the structure of the mucosa. The
physiology of the mucosa is similar to the skin. However there are
some notable differences:
[0365] the mucosa does not have a stratum corneum
[0366] the epithelium is 600-800 82 m thick. This is considerably
deeper than the epidermis of the skin (the exact thickness varies
with site, age and species).
[0367] Therefore, to target organelles within cells at the basement
membrane of the epithelium, a depth D.sub.c of 600-800 82 m is
required.
2.3 Lung Delivery
[0368] The epithelial cells on the lining of the lung are the
target for various gene therapy approaches. These cells are
underneath a mucous/liquid lining, which may be 10-100 82 m thick.
Therefore this lining needs to be overcome. In the example of gene
therapy for the treatment of Cystic Fibrosis, this lining is to be
overcome to target celial cells on the surface of the
epithelium.
2.4 Other Internal Organ Delivery
[0369] Cells within other the internal organs can also be targeted
for a range of applications, such as:
[0370] Internal cancers
[0371] Liver (e.g., for Malaria treatment) 1
[0372] Heart (e.g., for angiogenesis blood vessel formation).
2.5 In-Vitro Delivery
[0373] Tissue, cell-lines, tissue culture, excised organs, tissue
engineered constructs and artificial tissues may also be targeted,
in-vitro. Examples include:
[0374] stimulation of cell lines and cell monolayers in
culture.
[0375] stimulation/delivery of growth factors and/or genes (e.g.,
in tissue engineering and wound healing).
[0376] 3. Constructional Features of the Micro-Nanoprojections
[0377] 3.1 General Micro-Nanoprojection Dimensions
[0378] With typical cell and organelle scales described and
locations in tissues highlighted, the structural terms of the
nanoneedles individually are described, and then as examples of
arrays for targeting cells at different tissue sites.
[0379] Nanoneedles are configured to penetrate the cell membrane
with minimal damage, targeting the organelles of interest. The
overall dimensions of the nanoneedle projection are shown in FIG.
20. The nanoneedle is divided into two main sections:
3.1.1 The Targeting Section, Length (l)
[0380] Referring to FIG. 20, the radius of the tip (r) is to be as
small as practicable with limits set by manufacturing methods and
material considerations, where usually the diameter at the distal
end of l is d.sub.1. Usually di.about.2r. The diameter of the upper
end of l is defined as d.sub.2. Over the length l, the effective
diameter, tapering from d.sub.1 to d.sub.2 is to be considerably
less than the diameter of the cell (d.sub.cell) or other target. It
is shown in Table 1 approximately that 10 .mu.m<d.sub.cell<30
.mu.m. So, approximately, d.sub.1<<10 .mu.m, preferably <1
ideally in some cases <500 nm. Ideally, the scale of d.sub.1 is
to be of the order of the organelle of interest.
[0381] For practical engineering and manufacturing and purposes
(e.g., buckling/loading/fracture) it is often preferred that the
projection along l tapers out to a larger diameter
(d.sub.2>d.sub.1) that still is less than the diameter of the
cell (<d.sub.cell). However, along the length of l, the profile
may be configured such that the diameter is constant (i.e.,
d.sub.1=d.sub.2).
[0382] The length of the targeting section (l) is sufficient to
ensure that the organelle of interest is targeted (i.e.,
l>organelle dimension). For example, in targeting the cell
nucleus, l>3-6 .mu.m). Ideally, l is even longer to account for
variation in cell depth location (e.g., as shown by the variation
in the Langerhans cell depth Figure A) and increase the probability
of the desired needle-organelle contact. The upper length of l is
determined by the combination of material properties, needle shape
and loading to ensure that the needle does not break under
mechanical loading. Engineering analyses shows this mechanical
loading is mostly compression, with tension and bending moments.
Good engineering practice to ensure the material does not break
includes Euler buckling, fracture mechanics, and work-to-failure.
In considering a population of projections in an array, statistical
methods (e.g., Weibull statistics) and other related methods may be
applied to ensure a very small fraction of the projection
population breaks.
[0383] Consider, as an example, the case of compression loading
with the primary mode of projection failure is by buckling. This
compression buckling criteria set by the Euler Bucking force
(P.sub.cr)
P c .times. r = .pi. 2 .times. EI 4 .times. l 2 ##EQU00007##
where (E) is the Young's modulus of the nanoneedle material, I is
the moment of inertia (I=
.pi. .times. .times. d 4 64 ##EQU00008##
for a cylinder). Therefore the material properties of the
nanoneedle and shape determine the maximum force permitted for
buckling (P.sub.cr). Good engineering practice dictates that
P.sub.cr must be less than the insertion force of the needle, to
ensure it does not break.
3.1.2 Support Section of the Micro-Nanoprojection (a)
[0384] Referring again to FIG. 20, the support section of the
micro-nanoprojection (a) is sufficient to bring the targeting
section of the cell (l) into contact with the cells/organelles of
interest. The diameter of the support section along the length a
tapers at the distal end from d.sub.2 to d.sub.3 at the base, where
usually engineering and material considerations result in
d.sub.3>d.sub.2 to ensure it does not buckle or break by another
mode of failure. In some cases, the diameter along the length of a
may be constant (i.e., d.sub.3=d.sub.2). FIG. 20 shows that a, l
and the overall length of the projection (L) are related by:
L=a+l
[0385] In section 2 the depths of cells for various applications in
intradermal, mucosal, lung and internal organ delivery are
outlined. The rationale and approximate values of a for targeting
these individual sites are presented as examples.
[0386] In epidermal delivery, a is the sum of the desired depth in
the tissue and an allowance above the target (for instance for
tissue surface curvature). This dimension in epidermal delivery is
usually <200 .mu.m in length, and preferably <100 .mu.m in
length-depending on the tissue species and site. In the targeting
of dermal cells, this depth is even greater with a <1000 .mu.m,
and preferably <600 .mu.m. In another example, the targeting of
basal cells in the epithelium of the mucosa, an a of 600-800 .mu.m
is required. In lung delivery, the local bather of the mucous
lining to the cells is 50-100 .mu.m thus a would be of the order of
100 .mu.m in this case.
[0387] 3.2 Specific Applications
3.2.1 Example 1
[0388] Targeting the nuclei of LC in the viable epidermis, 40-50
.mu.m deep in the tissue. The force to insert the needle is
estimated with the Unified Penetration Model (proposed by Dehn J: A
unified theory of penetration. International Journal of Impact
Engineering, 5:239-248.1987).
F insert .apprxeq. 3 .times. A .times. .sigma. y = 3 .times. .pi. 4
.times. d 2 .times. .sigma. y ##EQU00009##
where .sigma..sub.y is the yield stress of the tissue.
[0389] Assume, in the highest loading case, the upper value of the
yield stress of the SC of 20 MPa applies to all the skin (from
Wildnauer R H, Bothwell J W, Douglas A B: Stratum corneum
properties I. influence of relative humidity on normal and
extracted stratum corneum. Journal of Investigative Dermatology,
56(1):72-78. 1971). Note, this estimate of insertion force is a
factor of 10 greater than calculations inferred from measurements
with a probe in the skin (unpublished results, by Crowley (2003,
4.sup.th Year Project, Engineering Science, University of
Oxford).
[0390] By setting F.sub.insert=P.sub.cr we arrive at
l = ( .pi. .times. .times. Ed 2 192 .times. .sigma. y ) 0.5
##EQU00010##
which approximates the Euler Buckling relationship between the
maximal permissible length of extension for a probe of diameter d,
constructed of a material with a Young's modulus (E), penetrating
into a tissue with a Yield Stress (.sigma..sub.y).
[0391] FIG. 21 plots the cell/organelle targeting section length
(l) as a function of diameter for six materials. Note that these
are upper limit calculations of the loading effects.
[0392] Consider the curve for titanium (E=116 GPa) in which a 400
nm diameter (d.sub.1=d.sub.2) cell/organelle targeting section
corresponds to a length of 7 82 m, and a 1 82 m diameter
corresponds to a maximum length of 14.1 .mu.m. In this case, the
nucleus of the LC is the organelle of interest with a size of 3-6
.mu.m. Here, we choose a organelle targeting section of
d.sub.1=d.sub.2=1 .mu.m and a length (l) of 10 .mu.m (symbols
corresponding to the diagram of FIG. 20). The support length (a) is
40 .mu.m, tapering to a base diameter (d.sub.3) of 5 82 m. These
dimensions are summarized in Table 2.
[0393] Alternatively, a nanoneedle constructed of a stiffer
material such as silicon (E=189 GPa) results in a smaller
cell/organelle targeting section of d.sub.1=d.sub.2=200 nm over a
length (l) of 5 82 m. Of course, a brittle material such as silicon
would also require fracture (and other) analyses that may lead to
more conservative (i.e., larger) dimensions-these numbers
throughout this section are simply illustrative.
[0394] Utility of an even stiffer material such as Tungsten (E=400
GPa) results in the same nanoneedle diameter of d.sub.1=d.sub.2=200
nm extended over a length (l) of >6 .mu.m-or alternatively the
possibility of a smaller diameter d.sub.1=d.sub.2<200 nm for a
length of 5 .mu.m.
3.2.2 Example 2
[0395] Targeting the Endoplasmic Reticulum LC of the Viable
Epidermis. Table 1 lists the thickness of the Endoplasmic Reticulum
envelope to be 75 nm. Choosing titanium, the organelle targeting
section is set at 400 nm over a length of 5 .mu.m. As shown in
Table 2, the remainder of the components are identical to those set
out in Example 1.
3.2.3 Example 3
[0396] Targeting cell nuclei at a depth of 600 82 m in the tissue.
Here, the targeting section is identical to Example 1, whereas the
considerably longer length (a=600 82 m) requires a significantly
wider base (d.sub.3=50 82 m).
3.2.4 Example 4
[0397] FIG. 20 shows an axi-symmetric design for the nanoneedle.
Alternative shapes may be used with:
[0398] higher values of mass-moment of inertia (I)
[0399] more knife-like geometries with sharper leading edges.
[0400] One possible example of an alternative shape taking on these
features is shown in plan view in FIG. 27.
TABLE-US-00003 TABLE 2 The geometry of three nanoneedle examples. r
d.sub.1 d.sub.2 l d.sub.3 a Example (nm) (nm) (nm) (.mu.m) (.mu.m)
(.mu.m) 1. Nucleus of LC, 40-50 .mu.m deep <1000 1000 1000 10 5
40 2. Endoplasmic Reticulum of <400 400 400 5 5 45 LC 40-50
.mu.m deep 3. Cell nuclei, 600 pm deep. <400 1000 1000 10 50
600
4. Route for Transfer of Bioactive Material to End Point
(Coating)
[0401] Consider the generic shape of the micro-nanoprojection (FIG.
20). The bioactive material is coated to the outer surface of the
micro-nanoprojection. The following lists two example protocols on
how this is done, followed by a list of possible coating
combinations.
4.1 Coating DNA on Metallic (e.g., Gold or Tungsten)
Micro-Nanoprojections
[0402] Consider the case of coating eGFP to tungsten
micro-nanoprojections. Essentially, published and well established
protocols devised for coating gold microcarriers for biolistic
delivery are employed. Examples of these protocols are detailed
in:
[0403] PowderJect and Bio-Rad patents and protocols such as:
http://plantsciences.montana.edu/wheat-transformation/biolisti.htm);
J. E. Biewenga, O. H. Destree, L. H. Schrama, "Plasmid-mediated
gene transfer in neurons using the biolistics technique", J.
Neurosci. Methods 71(1997) 67-75; S. Novakovic, M. Knezevic, R.
Golouh, B. Jezersek, "Transfection of mammalian cells by the
methods of receptor mediated gene transfer and particle
bombardment", J. Exp. Clin. Cancer Res. 18 (1999) 531-536; H.
Wellmann, B. Kaltschmidt, C. Kaltschmidt, "Optimized protocol for
biolistic transfection of brain slices and dissociated cultured
neurons with a hand-held gene gun," J. Neurosci. Methods 92 (1999)
55-64; and, John O'Brien, Sarah C. R. Lummis "An improved method of
preparing microcarriers for biolistic transfection", Brain Research
Protocols 10 (2002) 12-15.
[0404] In this example, subsequently applied to the Experimental
Exemplification 1 (section 5.3) the protocol of O'Brien and Lummis
(2002) is applied.
[0405] Alternatively, in coating silicon, protocols developed for
the sub-micron patterning of DNA oligonucleotides for
"lab-on-a-chip" technology can be applied to coating the
projections on the array (H. B. Yin, T. Brown, J. S. Wilkinson, R.
W. Eason and T. Melvin (2004) "Submicron patterning of DNA
oligonucleotides on silicon" Nucleic Acids Research, 2004, Vol. 32,
No. 14 el 18). This process is an example of two-stage process for
the covalent attachment of DNA oligonucleotides onto crystalline
silicon (100) surfaces. In summary, UV light exposure of a
hydrogen-terminated silicon (100) surface coated with alkenes
functionalized with N-hydroxysuccinimide ester groups result in the
covalent attachment of the alkene as a monolayer on the surface.
Submicron-scale patterning of surfaces is achieved by illumination
with an interference pattern obtained by the transmission of 248 nm
excimer laser light through a phase mask. The N-hydroxysuccinimide
ester surface act as a template for the subsequent covalent
attachment of aminohexyl-modified DNA oligonucleotides.
Oligonucleotide patterns, with feature sizes of 500 nm, are
reliably produced over large areas. The patterned surfaces are
characterized with atomic force microscopy, scanning electron
microscopy, epifluorescence microscopy and ellipsometry.
Complementary oligonucleotides are hybridized to the
surface-attached oligonucleotides with a density of
.about.7.times.10.sup.12 DNA oligonucleotides per square centimeter
(for example).
[0406] Some-possible coating combinations include:
[0407] No coating: The cell is perturbed/stimulated by the physical
disruption of the micro-nanoprojection structure. This physical
stimulus can be coupled with electric stimulus as a form of
specific nanoelectroporation of particular organelles or the
cell.
[0408] Coating entire nanoneedles: The bioactive material can coat
the whole surface of the nanoneedle (over the length L).
[0409] Selective coating: For certain applications only parts of
the nanoneedle need be coated. For instance, the targeting section
(l) can be selectively coated by only immersing this part of the
needle in the coating media. Alternatively, different combinations
of various coatings can be made on the one nanoneedle (combinations
of DNA and adjuvants, proteins, cytokines, inhibitors etc.). Or, in
the case of nanoneedle arrays, different coatings may be provided
on different individual micro-nanoprojections.
[0410] Coating materials: In addition to bioactive materials (such
as DNA, RNA and proteins), the micro-nanoprojections may also be
coated with nanobiosensors (e.g., quantum dots, nanomachines, MEMS)
using a range of coating protocols.
[0411] Material formulated in degradable micro-nanoprojections:
All, or part of the micro-nanoprojection can be constructed of a
biocompatible, biodegradable polymer (such as Poly Lactic Acid
(PLA), Poly Glucleic Acid (PGA) or PGLA), which is formulated with
the bioactive material of choice. The micro-nanoprojections may
then be inserted and, as they dissolve, the bioactive material will
enter the organelle(s)/cells.
[0412] In the cases of bonding of the biomaterials to the surface
of the micro-nanoprojections, access to the organelles may be by
the following:
[0413] The bioactive material can stay on the surface of the
nanoneedle, but elicit the response within the organelle/cell.
[0414] The bioactive material bonds can be broken by exposure of
enzymes, proteins or hydration within the cell/organelle.
5. Experimental Exemplification 1
[0415] This section outlines the simplest embodiment of the
nanoneedle concept: an individual projection, coated on the surface
with a bioactive material (here DNA coded for GFP to transfection),
used to deliver this material to a living cell, in-vitro, where a
biological response is measured (transfection) and the cell is
alive.
5.1 Fabrication of Micro-Nanoprojection
[0416] Following the engineering analyses discussed in section 3,
Tungsten was selected as the material for fabricating the
individual nanoprojection, largely because of its high stiffness
(E=400 GPa) and ease of fabrication.
[0417] Individual micro-nano projections were fabricated from
tungsten rod of diameter 280 .mu.m (Source, ADVENT Research
Materials Ltd) using electropolishing (approximately following the
protocol of Cerezo, A., Larson D. J. & Smith G. D. W. (2001)
"Progress in the atomic-scale analysis of materials with the
three-dimensional atom probe". Materials Research Society Bulletin,
26(2): 102-107.
[0418] The electropolishing set-up used a solution of distilled
water with 4% NaOH molar. For the electropolishing tungsten probes
with the sharpest tips, the following settings were used:
[0419] 3 mm of probe submersed into the solution
[0420] 10 volts A. C. for approx. 42 seconds.
[0421] Following optimization, electropolishing was ceased when
probe length began to diminish (detected by visual sighting by
naked human eye).
5.2 Example of Individual Electropolished Projections
[0422] Recall the dimension parameters correspond to the generic
micro-nanoprojection tip outlined in the schematic of FIG. 20.
[0423] FIGS. 22A-B shows Transmission Electron Microscopy (TEM) of
an electropolished Tungsten tip at two different resolutions, with
the scale bar in FIG. 22A of 20 .mu.m, and in FIG. 22B of 0.11
.mu.m. FIGS. 22B and 22C show the minimum tip size (d.sub.1) is
.about.100 nm (i.e., r.about.50 nm). By using the electropolishing
process, the projection gradually tapers to the full thickness of
the original rod (i.e., d.sub.3=280 .mu.m). In this case, the cell
targeting length (l) is approximately 20 .mu.m-defined by where the
diameter (d.sub.2) remains less than .about.1 .mu.m.
[0424] Individual micro-nano projections were glued into hole
drilled into perspex cylindrical holders to allow fitting into
microprojection systems (see section 5.5).
5.3 Coating of Micro-Nanoprojections with eGFP DNA Plasmid
[0425] The tungsten micro-nanoprojections were coated by adapting a
protocol devised for the coating of gold microparticles for
biolistic delivery developed by O'Brien and Lummis (2002) "An
improved method of preparing microcarriers for biolistic
transfection", Brain Research Protocols 10 (2002) 12-15. The
adapted protocol is as follows:
[0426] Coat tungsten micro-nanoprojection in 50% glycerol/50% water
solution by dipping the wire in the solution for 10 minutes.
[0427] (ii) Remove the tungsten.
[0428] (iii) Individual tungsten micro-nanoprojections were placed
in a tube to which was added 50 ml of 1 M spermidine for 5
minutes.
[0429] (iv) Add 1 .mu.g/ml of 50 ml of DNA Spermidine and gently
mix the two solutions with aspiration with the wire in the
solution.
[0430] (v) Gently, the tube was stirred for a few minutes, and
then, whilst stirring, 1 M CaCl in solution was added in a drop
wise manner.
[0431] (vi) Gently mix the solution and let DNA precipitate on to
the wire for 30 minutes.
[0432] (vii) Remove wire from solution and dip it in 100% ethanol.
Repeat this process 3 times.
[0433] (viii) Let the wire dry before use.
[0434] (ix) The process was then repeated for the other
micro-nanoprojections.
[0435] FIGS. 23A and 23B show images of lengths of tungsten wire
(280 82 m diameter) imaged with a fluorescent microscope with
uncoated wire in FIG. 23A and DNA coated wire (using the described
protocol, above) immersed in a liquid with a propidium iodide stain
(red fluorescing dye) in FIG. 23B. As expected there is no red
fluorescence with the uncoated wire, while the strong red
fluorescence of the NDA coated tungsten qualitatively shows that
the coating was successful.
5.4 Assessment of Delivery into In-Vitro Test Bed
[0436] To assess the delivery profile of DNA in the in-vitro
equivalent of skin tissue, DNA coated projections were inserted
into an agar (3%) gel stained with acridine orange (green
fluorescing dye). This gel (without the stain) is routinely used
for in-vitro testing of biolistic to devices for DNA vaccination
and other applications.
[0437] The uncoated and coated tungsten rods were inserted and
removed by hand into the agar (to a depth >1 mm) for a cycle
duration of approximately 1 second.
[0438] FIGS. 24A-24D show optically sectioned Multi-Photon
Microscopy (MPM) images of the agar after insertion of a DNA coated
tungsten probe on the surface in FIG. 24A, at a depth of 13 .mu.m
in FIG. 24B, at a depth of (32 82 m) in FIG. 24C. FIG. 24D shows an
optical section at 32 82 m of agar gel following insertion of a
probe without a DNA coating.
[0439] On the surface of the agar, there is very little
fluorescence, indicating little DNA. In contrast, at depth sections
of 13 .mu.m and 32 82 m the fluorescence in the coated case is
significant-indicating that DNA remained intact on the surface of
the probe during insertion and then came off by exposure of the gel
and/or by the action of removing the probe. This fluorescent signal
was very strong compared with the uncoated probe control at 32 82
m.
5.5 Experiment of DNA Delivery Into a Cell
[0440] The described electropolished tungsten micro-nanoprojections
coated with GFP plasmid DNA were tested, with the objective of
determining whether the DNA could be delivered to transfect the
cell, without damaging the cell.
[0441] The cell line is called A549 which is a human lung carcinoma
(epithelial) cell line. Cells were cultured in Dulbecco's modified
eagle medium (DMEM) supplemented with 10% fetal calf serum at
37.degree. C., 5% CO.sub.2.
[0442] These cells were mounted on three separate Petri dishes with
an optical slide underneath. The adherent properties of the cells
ensured they remained in contact with the base of the dish.
[0443] One of the Petri dishes with cells was set aside as a
control. The other two Petri dishes were probed with the
micro-nanoprojections. In total, 6 micro-nanoprojection systems
were used.
[0444] Probing of the cells was performed with a microinjection
system (Eppendorf Femtoject and Injectman NI2 microinjection kit)
fitted to an inverted fluorescent microscope (Zeiss Axiovert 25
microscope with stage heater).
[0445] The micro-nanoprojection were fitted into the standard
microinjection holder and then moved with course and fine
adjustment of a motorized X-Y-Z axis controller to just contact the
cell membrane, before retraction of less than 1 82 m.
[0446] A cycle time of probing was set between limits of 0.1 and 15
seconds, with a traverse distance of 3-5 .mu.m-then the cycle was
automatically performed on the system.
[0447] After 3 cells, the probe was replaced and refitted with a
replacement. In total 10 cells were perturbed with a
micro-nanoprojection coated in eGFP plasmid DNA.
[0448] All the cell samples were then incubated for 24 hours before
viewing with a wide field fluorescent microscope, with the
appropriate fluorescent filters. The observed results show: in the
control there is no sign of eGFP transfection, as expected.
However, in the cell micro-nanoprojection cases, we see that 10
cells have transfected. Thus we have a 100% transfection efficiency
and no evidence of cell death.
[0449] These data prove the micro-nanoprojections, coated in DNA
can individually target cells, transfecting them and not kill
them.
[0450] Also, subsequent analysis of the micro-nanoprojections
showed they remained intact, illustrating that unlike biolistic
delivery, the "carrier" material is not left behind.
6. Experimental Exemplification 2
[0451] With the biological result achieved on a single cell,
in-vitro (Experimental exemplification 1). this section outlines a
logical progression, which is the physical testing of an individual
micro-nano projection into a representative skin sample for
structural integrity-thereby testing the engineering analyses
outlined in section 3.
6.1 Experiment Design
[0452] Using the electroporation process described in Embodiment 1,
a micro-nanoproprojection was fabricated of Tungsten with a tip
radius (r) of <400 nm.
[0453] The tissue was freshly excised Balb/C mice ears (age 8-10
weeks), which were glued to a metal cylinder holder. The reported
stratum corneum and viable epidermis thicknesses of these samples
are respectively .about.5 82 m and 12 82 m (Arbuthnott (2003)).
[0454] The tissue was indented by fitting the micro-nanoprojection
to a Nanoindenter (MTS systems, UK), measuring a force displacement
curve. FIG. 26 shows two typical sample results of
loading-unloading curves achieved to a depth of .about.50 .mu.m.
These results show that the micro-nanoprojection probe penetrated
through the epidermis and well into the dermis without damage. The
difference in magnitude between the curves is typical of biological
variability observed in bio-viscoelastic tissue. Importantly,
unlike ballistic particle delivery, the depth of penetration is not
dependent upon this variability-rather the length of
projection.
[0455] Indeed, the projections did not break-the loading experiment
was simply stopped at a 50 82 m setting on the experimental
apparatus. This was confirmed by imaging with projections with a
microscope after the experiment. This experiment was repeated 20
times, confirming the engineering analyses of probe structure
required to target cells in the skin is applicable and valid.
7. Experimental Exemplification 3: Overall Size Range of Individual
Patch
[0456] With the biological and engineering criteria investigated on
an individual projection, the next logical step is to extend the
concept to arrays of patches targeting tissue sites. Presented are
several patch arrangements, each designed for a particular
targeting need of cells, organelles and/or tissue sites.
7.1 General Dimensions and Design of Patch
[0457] FIG. 28 shows a schematic of an array of the described
micro-nanoprojections configured on a patch, with the spacing
between micro-nanoprojections defined as (S) and the patch breadth
defined as (B). Recall, the basic dimensions and definitions of an
individual micro-nanoprojection are outlined in section 3 and shown
schematically in FIG. 20; these are referred to again here.
[0458] Operation of the patch with the micro-nanoprojections is,
for example:
[0459] (a) the slow insertion, by hand (or other means, such as a
spring) of the patch onto the tissue, with the
micro-nanoprojections inserting into the target tissue of
interest.
[0460] (b) the patch including the micro-nanoprojections are held
in place for a sufficient time for the "event" (biological,
physical or other) to take place. This may be instantaneous, or in
other cases could take days, weeks or months.
[0461] (c) the patch including the micro-nanoprojections are then
retracted.
[0462] These three stages form a cycle, that may be operated by
hand or automated with the aid of suitable mechanical, electrical
or electro-mechanical devices.
[0463] Patches may be applied to the tissue site once, or a
multitude of times depending upon the effect desired.
[0464] The case examples presented in 7.2 all center on the
targeting of Langerhans cells or organelles within these cells and
the overall dimensions of the individual micro-nanoprojections are
configured, using the approximate guideline of Example 1 and 2
shown in Table 2. However, as Table 2 also shows, these parameters
(r, d.sub.1, d.sub.2, d.sub.3, a) can vary significantly, depending
on the cell/organelle and its position relative to the tissue
surface.
7.2 Case Examples of Patch Configurations
[0465] The overall size (B.times.B) of the patch and spacing (S)
between the micro-nanoprojections is determined by the application.
Note, in all cases, the patch could be square (B.times.B),
circular, elliptic, or any other suitable shape. For simplicity,
the square dimensions are quoted throughout. Generally the size is
to be less than 15.times.15 mm (B), with 1<S<1000 .mu.m,
preferably with 10<S<100 .mu.m.
[0466] Note that an alternative for these and other patch examples
is for larger-patches, giving amplified responses and/or which may
be easier to handle having regard to end
users/patients/practitioners.
7.2.1 Targeting LC Nuclei
[0467] Consider Example 1 from Table 2, which is the targeting of
nuclei of Langerhans cells. Kendall et al. (2003); Kendall M. A.
F., Mulholland W. J., Tirlapur U. K., Arbuthnott E. S., and
Armitage, M. "Targeted delivery of micro-particles to epithelial
cells for immunotherapy and vaccines: an experimental and
probabilistic study", The 6.sup.th International Conference on
Cellular Engineering, Sydney, August 20-22), suggest that to
trigger cellular responses in DNA vaccination, of the order of 100
nuclei of LC are effectively transfected in gene gun applications
that lead to the desired cellular (MHC 1) systemic response. A
probability analysis has been performed to determine the
configuration of patch required to transfect 100 cells. The
probability of one micro-nanoprojection making contact with an
organelle (or cell) defined over an area and volume is set by:
P contact = V probe V layer V organelles V layer ##EQU00011##
[0468] In the analysis, it is assumed:
[0469] that there is no cell death.
[0470] the cell/organelle targeting sections (l) are configured to
be at the correct depth (i.e., the depth of the Langerhans cells),
and, for simplicity, parallel and 1im in diameter
(d.sub.1=d.sub.2=1 .mu.m).
[0471] the cell/organelle targeting sections (l) are coated in DNA.
Of course, for simplicity of coating, it is possible that most or
all of the micro-nanoprojection structure is coated in DNA.
[0472] contact of any part of the micro-nanoprojection along the
cell/organelle targeting section with the nucleus is the only mode
that leads to transfection (i.e., cytoplasm delivery, cross-priming
and other modes are ignored).
[0473] With a 1 .mu.m diameter cell/organelle targeting section
(1), the probability of contact with a LC nucleus is 0.0131. This
probability is .about.1000-1500 times higher than a typical
probability of a direct "hit" with the biolistics approach and
microparticles. Furthermore, the transfection comparison is even
more favorable for the micro-nanoprojection patch, given far fewer
cells are killed than by the gene gun.
[0474] Table 3 is a summary of configurations of patches that may
be used. At one end of the range, a spacing of 100 rods/mm.sup.2
corresponds to a patch surface area of 76 mm.sup.2, with a spacing
(S) of 100 82 m, a breadth of 8.7 mm and a total number of rods of
over 7000. Increasing density of rods to 1000/nm.sup.2 results in a
reduction in patch size to less than 3 mm.times.3 mm.
TABLE-US-00004 TABLE 3 Calculated Patch Configurations for the
Targeting of Nuclei of LC. Area of patch Spacing (S) Breadth of
Rod/mm.sup.2 (mm.sup.2) (.mu.m) patch (B) (.mu.m) Total Rods 100
76.4 100 8.7 7639 500 15.3 45 3.9 7639 1000 7.6 32 2.8 7639
7.2.2 Targeting the LC
[0475] In Table 4, this analysis is extended to the configuration
of patch required to target 100 LC (i.e., anywhere within the
complete cell), using micro-nanoprojections with the same geometry
as above, with the probability of the event 0.063. Not
surprisingly, this is higher to than the probability of targeting
the nuclei. Hence, fewer rods are needed and the patch size can be
smaller (4.times.4 mm down to 1.25.times.1.25 mm).
TABLE-US-00005 TABLE 4 Calculated Patch Configurations for the
Targeting of Anywhere Within Complete LC. The Nanoneedle Diameter
d1 Is Assumed to Be 1 .mu.m. Area of patch Spacing (S) Breadth of
Rod/mm.sup.2 (mm.sup.2) (.mu.m) patch (B) (.mu.m) Total Rods 100
15.8 100 4.0 1578 500 3.2 45 1.8 1578 1000 1.5 32 1.3 1578
7.2.3 Targeting the Endoplasmic Reticulum of the LC
[0476] In Table 5, the analysis is also applied to the targeting of
the Endoplasmic Reticulum (Example 2 from Table 2), which may be
required for targeted RNA delivery. Using the assumptions from
above, with a probe diameter of 400 nm the probability of a single
needle contacting the Endoplasmic Reticulum is 0.031. In this case,
the size of patch ranges from 8.times.8 mm to 2.5.times.2.5 mm
TABLE-US-00006 TABLE 5 Calculated Patch Configurations for the
Targeting of the Endoplasmic Reticulum of LC. The Nanoneedle
Diameter Is Assumed to be 400 nm Area of patch Spacing (S) Breadth
of Rod/mm.sup.2 (mm.sup.2) (.mu.m) patch (B) (.mu.m) Total Rods 100
65 100 8.0 6451 500 13 45 3.60 6451 1000 6.5 32 2.54 6451
7.2.4 A Combination of Micro-Nanoprojection Geometries on a
Patch
[0477] A patch may also have combinations of micro-nanoprojections,
with different geometries. For example the length (L) or indeed the
other parameters in FIG. 20 may be varied throughout the patch,
either in defined sequences, clusters and/or randomly (within
limits). Within this, or separately, the diameter of the projection
may be increased on individual micro-nanoprojections significantly
in order to induce cell death at controlled locations. This, for
example, may be used to induce bystander biological responses
(e.g., stimulation/inflammation/activation) to neighboring healthy
cells-which could have or will be interacting with described,
smaller micro-nanoprojections configured for minimal cell
damage.
8. Specific Bioactives, e.g., Nucleic Acids
[0478] The bioactive or other stimulus is to be coated or part of
the nanoneedle array. The choice of bioactive is determined by the
application and target organelles or cells of interest. This range
encompasses, but is not restricted to:
[0479] No coating
[0480] Polynucleotides, DNA (all variants), RNA (all variants),
proteins, antigens, allergens and adjuvants, molecules,
compounds.
[0481] Biosensor molecules and compounds and materials.
[0482] Nanosensors (MEMS etc.).
[0483] Combinations of the above, on a
9. Methods of Production of Device
[0484] General requirements of the manufacturing method are:
[0485] a radial resolution of <200 nm;
[0486] to construct the nanoneedle array on a patch (e.g., FIG. 19)
in a scaleable process for high throughput manufacturing with
minimal human input.
[0487] Construction to be of a medical grade material (e.g., Gold,
Silver, Titanium, Tungsten or PLA, PGA, PGLA, Silicon)
[0488] A range of techniques for micro-nanofabrication methods
described in text books, papers and in other literature (e.g.,
Madou M. J. "Fundamentals of Microfabrication. The science of
miniaturization", CRC Press, 2002; McAllister et al. 2003, PNAS,
Nov. 25, 2003, Vol 100, number 4) are applicable to the nanoneedle
device here.
[0489] Furthermore there are a range of materials and fabrication
methods being developed that also have potential utility as part of
a micro-nanoprojection array. Three construction embodiments are
shown here as examples.
[0490] In these constructional embodiment example cases, the patch
geometry of manufacture is summarized in Table 3, with an area of
patch .about.76.4 mm.sup.2, a spacing of 100 .mu.m and the minimum
tip size of 1 82 m (d.sub.1). A schematic of this array on patch is
shown in FIG. 29. Of course, the techniques can be applied to a
range of geometries.
9.1 Constructional Embodiment Example 1
[0491] As an example of the fabrication of silicon
micro-nanoprojections, the following is applied. Deep Reactive Ion
Etching (DRIE) is used as a process ideal for these high aspect
ratio structures, where in one example (Oxford Instruments
Plasmalab System 100, Modular ICP180 Etch System-S12), etch rates
of >2.5 .mu.m/min, and sometimes >5 82 m/min, are possible.
Full details of etch protocols for this system are available in the
literature, including Oxford Instruments company manuals.
[0492] Briefly, chromium was sputter deposited then
lithographically patterned as an array of dots onto 3 inch (75 mm)
100 oriented silicon wafers. The array of dots had a
center-to-center spacing of 100 .mu.m, and in some earlier test
cases, 10-20 .mu.m. Similarly, through iteration with the
conditions and exploring the extent of undercut, the diameter of
the dots 1 82 m, and in other cases, 3 82 m, 5 82 m, and 10 .mu.m.
Deep Reactive Ion Etching (DRIE, Oxford Instruments, Bristol, UK)
was then carried out to obtain the desired by profile by adjusting
the etch rate.
[0493] The typical instrument conditions included a 20 standard
cm.sup.3/min (sccm) SF.sub.6 and 15 sccm (O.sub.2) at a pressure of
20 Pa. Power was varied. The etching process was performed at
cryo-cooled conditions, achieving temperatures of -100 to
-150.degree. C. Lower etch rates were used for the tapered
sections, whereas higher rates were used for the more parallel
sections. Micro-nanoprojection fabrication was finished when the
chromium masks became fully undercut and fell off the projection
tips. The process is completed in less than 100 minutes, resulting
in several patch configurations on the wafer.
[0494] The patches were then cut from the silicon wafer, with a
dedicated cutter.
[0495] To increase the strength of the base of the patch, an
additional, thicker material is affixed to the back surface (i.e.,
the surface without the projections).
[0496] This process is repeated to fabricate a large quantity of
micro-nanoprojection array patches.
[0497] A variation of the described DRIE method may also be applied
to other materials, including Silicon Carbide (SiC).
9.2 Constructional Embodiment Example 2
[0498] This constructional embodiment example applies to a broader
range of micro-nanoprojections materials than those described in
the constructional embodiment Example 1. These materials include
Metals, polymers, silicon and oxides/carbides.
[0499] As an example, this case illustrates a method of fabricating
tungsten micro-nanoprojection arrays on a patch. Three of the steps
in this production process are discussed.
[0500] Step 1. Construction of a Template (Male)
[0501] To construct a male template with the profile of the
micro-nanoprojections, LIGA is used (a German acronym for X-ray
lithography, electrodeposition and molding). This template will
serve in the production of several molds in a soft polymer. LIGA,
which utilizes a Synchroton, is ideal for creating the template
because it has a very high resolution (<20 nm), can fabricate in
metals, and will easily fabricate a patch (maximum component size
is 3.4 inches in the Axsun technologies system). Fabrication
protocols to construct the template are detailed in Chapter 6 of
Madou M. J. "Fundamentals of Microfabrication. The science of
miniaturization", CRC Press, 2002 manuals and literature from
companies (e.g., AXSUN Technologies, Ca, USA). The choice of
materials for fabrication include Nickel (Ni), Nickel-Iron (NiFe),
Nickel-Cobalt (Ni--Co) Gold, Copper and Silver. In this example,
Nickel is selected as the material from which the
micro-nanoprojection array template is constructed.
[0502] It should be noted here that as an alternative, LIGA could
be used to make individual patches directly (e.g., of gold or
silver-which are both biocompatible) in large quantities. With
current costs of the LIGA technology, this is not practical-but
with LIGA technology advances, this could be feasible in the
future.
[0503] Step 2: Construction of a Mask (Female Component)
[0504] Because of the discussed current characteristics of LIGA
(e.g., cost), LIGA is not used to mass-fabricate
micro-nanoprojection array patches for direct use with the tissue
in large numbers. However, here it can be used as a template for
many multi-stage processes. As one example, the template is used
for multiple insertions into a soft polymer to produce a mask, as
shown in FIGS. 30A-30C, performed in the following steps:
[0505] FIG. 30A Insertion of the template directly into the soft
polymer. This is repeated several times to produce many
indentations in the polymer with a given mask.
[0506] FIG. 30B Allowing the soft polymer to harden, or "cure" with
the addition of catalysts and or temperature.
[0507] FIG. 30C Remove the template to leave behind a mask.
[0508] Step 3: Final Construction of the Nanoneedle Array with the
Mask
[0509] The masks are then be placed in a vacuum chamber and
deposited with a vacuum deposition/sputtering process. The material
to be sputtered is a biocompatible inert material, such as
titanium, gold, or silver. In this process, the polymer surface is
treated with an air discharge before the chamber is pumped down to
a vacuum at 27 degrees C. The titanium, gold or silver film is then
deposited. Commercial sputtering machines may be used in this
process (such as the VarianVM8 Sputterer).
[0510] If a charge is required to produce an anode to enhance the
coating of the nanoneedle section, then the end extension of this
piece can be "opened up" to expose a stronger anode which the
positive charged metallic ions in solution are attracted to. This
technique makes use of Magnetron Sputtering.
[0511] Step 4: Constructional Embodiment Example 3 Method
[0512] The mask can then be removed by immersion of a liquid (e.g.,
alcohols) to dissolve it.
9.3 Constructional Embodiment Example 3
[0513] Alternatively, a mask (female) can be directly manufactured
using Two-Photon Stereo Lithography (2PSL), in which the geometry
co-ordinates shown in FIG. 30(C) are applied to construct the
desired shape with the photosensitive resin. Femtosectond
two-photon stereo-lithography (2PSL) is described in detail by Miwa
et al. (2001, Appl Physics. A 73, 561-566). Zhou et al. (2002, Vol
296, Science), Stellacci et al. (2002, Adv. Mater. 14 (3)) and
Halik et al. (2003, Chem Commun 1490-1491). This to approach has
demonstrated the ability to construct complicated 3D shapes with a
resolution of <200 nm out of materials including photosensitive
polymer resins and metals in conjunction with dyes. Lattice
structures of the desired scale have been constructed with the
technique using resins impregnated with Silver (from Stellacci et
al. 2002).
[0514] Briefly, the technique works by scanning with a femtosecond
laser in a liquid photosensitive resin bath. The two-photon effect
ensures that solidification occurs only where the energy of the
laser is concentrated to a femtolitre volume (typically 200
nm.times.200 nm.times.400 nm, x, y, z). The method of manufacture
is to start at the tip of the nanoneedle (marked A in FIG. 29) and
then work up the needle to the base.
[0515] The same approach applies to the rest of the nanoneedles.
The process is fully automated with a motorized x-y stage with the
laser co-ordinates determined from engineering drawings of the
structure. Therefore, thousands of nanoneedles can be constructed
in a scaleable process. The technique allows complicated 3D
structures to be constructed by the scanning of a pulsed (80 MHz),
femtosecond pulse length laser concentrating light at 400-1000 nm
to a femtolitre volume to induce a two photon excitation of the
material and induce solidification.
[0516] The nanoneedles may be constructed with a photosensitive
polymer/resin, (such as the commercial grade SCR 500) or an
alternative impregnated with Silver (following Stellacci et al.
2002).
[0517] These materials are currently not suited to be used directly
in the skin due to insufficient stiffness. Young's Modulus of SCR
500 is 0.49 Gpa compared with
[0518] 116 Gpa for Titanium or 77.2 Gpa for Gold.
[0519] Not medical grade material. At the time of writing,
photosensitive resins such as SCR 500 had not gained approval for
medical grade purposes.
[0520] However, the technique is rapidly improving and may be a
possible fabrication method in the future.
9.4 Constructional Embodiment Example 4
[0521] In this case nanoneedles are constructed with silicon, with
a 200 nm tip for a length of 5 .mu.m followed by two other parallel
sections separated by tapered sections. This structure may be
constructed by Electron Beam Lithography and Reactive Ion Etching,
a standard technique in to the microelectronics industry. The
manufacturing approach has been applied by Henry et al. (Journal of
Pharmaceutical Sciences 87(8), 1998) and in Lebouitz and Pisano et
al. (U.S. Pat. No. 5,928,207) in the construction of
microneeedles.
[0522] FIG. 31 shows the structure produced with one of these
techniques. In this case of the nanoneedles, each parallel section
may be constructed with silicon 100 wafers and the tapered sections
constructed with silicon 111 (or other) wafer material-where the
angle of the taper is a preferred etching line of the material. The
shown shape is made from 5 wafers (one wafer per geometry). A
gradual taper mold can be constructed from the one silicon
wafer.
[0523] Alternatively, the silicon arrays may be used as masters for
microarray molds.
10. Methods of Treatment
[0524] Recall in section 1 the specific target sites of key cells
and organelles are described, followed in section 2 by their
location within many tissue and in-vitro sites. With the described
approach of targeting individual cells, then several cells and the
shape/coating/fabrication considerations outlined, the methods of
applying these micro-nanoprojection arrays to various tissue sites
are now discussed.
10.1 Intradermal Application
[0525] With the described embodiments for the skin previously
described, the patch is inserted into the skin either by user
control or by mechanical, electrical or other controlled means. All
these options are possible, as the magnitude of the forces is low.
For example, the insertion force is approximately calculated to be
less than 1 N (based on 7000 probes, 1 82 m in diameter piercing
tissue with a yield of 20 MPa).
[0526] Similarly, the time of insertion, residence and removal from
the tissue can be controlled by user and/or the described
mechanical/electrical means.
[0527] One embodiment of a simple application system for the patch
is shown in FIGS. 32A and 32B.
[0528] In this example, the application system 3300 is formed from
a structure in the form of a housing or body 3310. The body 3310
defines a cavity 3320 having an opening 3330. In use, a patch 100,
having a number of projections 110 provided on a base 120, is
moveably mounted within the cavity 3320. This may be achieved in
any one of a number of manners, to but typically involves having
the patch 100 suitable sized to allow movement along the cavity
towards and away from the opening 3330.
[0529] The patch 100 is mounted to an actuator, so that in use the
patch may be moved from a retracted position shown in FIG. 32A, to
an extended position shown in 32B. The actuator may be of any
suitable form, but in one example, includes a spring 3340 and a
releasing means 3350.
[0530] In the retracted position, the spring 3340 is biased against
the patch 100, thereby urging the patch 100 towards the opening
3330, with the patch 100 being retained in the retracted position
by the releasing member 3350. When the releasing means is
activated, for example, by having an operator release the wire, the
spring 3340 urges the patch 100 towards the opening 3330, so that
the projections 110 extend therethrough.
[0531] It will be appreciated from this that if the structure 3310
is positioned within the opening 3330 adjacent a subject's skin
3360, then operation of the releasing means 3350 causes the patch
100 to be pressed against the subject's skin 3360, so that the
projections 110 enter the viable epidermis as described above.
[0532] In the retracted position (FIG. 32(a)) the
micro-nanoprojections patch is recessed and thus protected,
preventing accidental administration. Other packaging/devices may
also be used for these purposes, for example a protective cap to be
removed before administration.
[0533] In this example, the patch is attached to a compressed
spring that is held in place by a tensioned string. It will be
appreciated that the releasing means typically is arranged so that
the patch 100 is retained in the retracted position until the
releasing means is activated. Thus, the patch 100 may be retained
in position by a clip that is released upon activation of the
releasing means. This allows the operator to position the device
against the subject's skin 3360 without having to retain the patch
in the retracted position. When located on the tissue, the string.
is released by a means (e.g., pressing a button, not shown in FIG.
32) and the patch is released to enter the tissue surface (FIG.
32(b)).
10.2 Mucosal Application
[0534] To effectively target mucosal sites (e.g., mouth, nasal,
rectal, vaginal), the described patch arrangements may be fitted to
an applicator designed to safely transport the patch into the mouth
and to accurately locate on to the mucosal site. This could be done
by using the described intradermal patch in the mouth.
[0535] Alternatively, FIG. 33 shows one example of and applicator
for mucosal delivery for the buccal mucosa (mouth). In this
example, the patch arrangement from FIG. 32 in the form of the
application system 3300 is coupled to an arm 3400, allowing the
application system 3300 to be positioned in otherwise hard to reach
places. The spring arrangement allows the patch to be applied, but
other mechanisms equally could be used. The applicator could have a
button at the distal end of the arm 3400 from the patch to allow
the operator to release the patch onto the mucosal site.
[0536] Other features may include a knurled surface towards the
distal end to aid user grip.
10.3 Lower Airway/Lung Application
[0537] FIG. 34 shows a schematic of the physiology of the airways
in a human. To apply the micro-nanoprojections to targeting cells
in the tracheal or lung lining, an applicator that can flexibly and
compactly reach these sites, target the sites, and then retract, is
required.
[0538] As one example, FIGS. 35A-35C show a deployable structure
embodiment for targeting these sites.
[0539] In this example, the apparatus includes an application
system 3600 mounted to a flexible structure, such as an arm,
allowing the application system 3600 to be inserted into a
passageway 3620, such as an airway, of the subject. To allow the
application system 3600 to be guided to the correct location, the
flexible structure may be in the form of a manipulable fiberscope,
or the like.
[0540] The application system includes a structure such as a body
3630, having a number of arms 3640 movably mounted thereto, to
allow the arms to move between a retracted position shown in FIG.
35B and an extended position shown in FIG. 35C. In use, the arms
3640 are typically biased towards the extended position, and are
retained in the retracted position, using a releasing mechanism,
such as a clip or the like. It will be appreciated that this may be
similar to the releasing means described above with respect to FIG.
34.
[0541] In any event, patches 100 are mounted to the arms, so that
when the arms are released, the patches are urged against a surface
3621 of the passageway 3620, allowing the projections 110 provided
on each patch to penetrate the surface 3621, and deliver material
or stimulus to prospective targets.
[0542] In the example shown, the eight arms 3640 are provided in
two sets of four, with the four arms in each set being
circumferentially spaced around the body 3630, as shown in FIG.
35C.
[0543] The method of operation is as follows. The flexible
structure (FIG. 35A) is guided through the throat to the site of
interest. This device may be fitted with imaging/illumination
systems to help guidance. FIG. 35B shows the system in location in
a "retracted" position. The arms fitted with the patches are held
in place against a spring load with pivot points. Then, by
mechanical actuation, the arms are released (FIG. 35C), with the
springs providing the force for location on the tissue.
[0544] The device does not need to be exactly centralized as the
arms are self-locating. By tension with wires, the arms are
retracted, and the device is removed through the throat.
[0545] In another embodiment, the arms are replaced by an
inflatable structure (a "sock") fully coated by the
micro-nanoprojections on the outside surface. When deflated, this
sock would be held in a structure which would look similar to FIG.
35A on the outside. When in place, the sock would be inflated via a
pressurized gas, locating on the tissue wall. This approach would
be particularly useful where large surfaces need targeting, such as
the lung. At the required time, the pressure is released with a
valving arrangement and the sock collapses back into flexible
targeting system and the device is removed.
10.4 Other Internal Tissues
[0546] Other internal organs or tissues (e.g., liver, kidney,
heart) are not as readily accessed as those described above. Here,
more invasive means are required to expose the site of interest
before targeting. One example is a more compact "catheter" version
of the described lower airway/lung targeting devices, reaching the
site via keyhole routes.
[0547] Another embodiment is surgery to fully open the site before
application with the patch.
10.5 In-Vitro Sites
[0548] The micro-nanoprojections may be fitted to patches for
in-vitro targeting, allowing a high-throughput targeting of cells.
In one example, larger patches with thousands and perhaps millions
of projections could be mechanically lowered onto cell monolayers,
and then removed, similar to a mechanical "press" arrangement. This
could, for instance, allow a mass transfection of cells,
in-vitro.
[0549] As another variation, cells are dynamically moving in a
shallow fluid stream. As these cells move, they pass below large
plates with thousands, and perhaps millions of
micro-nanoprojections. These plates pressing into this cell layer
repeatably, in a synchronized manner so that each pressing cycle
targets the appropriate batch of cells.
* * * * *
References