Professional Documents
Culture Documents
2006 1:39pm
CONTENTS
18.1 INTRODUCTION
The skin represents an attractive gateway for the localized and systemic delivery of therapeut-
ically active molecules due to its ready accessibility, avoidance of gastrointestinal degradation
and liver inactivation, monitoring capability and potential for improved patient compliance.
The ability to deliver therapeutic quantities of medicaments to and through the skin, however,
is dependent on the physicochemical properties of the candidate drug and the significant barrier
properties of the target tissue. After all, a primary function of the skin is to restrict the ingress of
external matter through physical blockade and immune surveillance. Whereas traditional
transdermal delivery techniques use formulation strategies to promote the transport of small
molecules through the stratum corneum, a growing number of delivery techniques that aim to
bypass or disrupt the skin barrier have been developed. Such strategies, including the use of
chemical enhancers [1], iontophoresis [2], electroporation [3], and sonophoresis [4] may sup-
plement traditional transdermal delivery strategies or provide a means of delivery for new drug
candidates, including macromolecules. Despite these advances, and a few exceptions, it could
be argued that effective transdermal delivery is still generally restricted to a small number of low
molecular weight, weakly lipophilic, and potent therapeutic molecules. Increasing emphasis on
337
Elka Touitou / Enhancement in Drug Delivery 3203_C018 Final Proof page 338 3.10.2006 1:39pm
bypass the stratum corneum in a more sophisticated manner with an associated reduction in
pain and diminished risk of phlebitis, hematoma, and thrombosis. Although modern fabri-
cation methods are now used to manufacture individual needles, this chapter will focus on the
innovative new technologies at the forefront of intra- and transdermal drug delivery.
(a)
Stratum corneum
Epidermis
Dermis
Pain receptors
Blood vessels
(b) (c)
FIGURE 18.1 Schematic representation of the concept for microneedle-assisted delivery. (a) The micro-
needles penetrate the stratum corneum, to facilitate access of molecules to the viable epidermis, without
impacting on the underlying nerve endings and blood vessels; (b) when removed the microneedles have
created conduits for drug delivery; (c) hollow microneedles allow direct injection of the formulation.
Elka Touitou / Enhancement in Drug Delivery 3203_C018 Final Proof page 340 3.10.2006 1:39pm
impinge on the nerve fibers and blood vessels that reside primarily in the dermal layer. The
micron-sized channels can therefore facilitate the delivery of both small and large molecular
weight therapeutics into skin without causing pain or bleeding at the site of application [10].
The advantages of using microneedles include: (a) direct and controlled delivery of the
medicament, (b) rapid exposure of large surface areas of epidermis to the delivery agents
(microneedle arrays can contain over 1000 microneedles), (c) effortless, convenient, and
painless delivery for the patient, (d) ability to manipulate the drug formulation, e.g., solution,
suspension, emulsion, dry powder, and gel, (e) enhancing the impact of concomitant delivery
methods such as transdermal patches, and (f) a minimally invasive methodology suited to
patient self-administration without the need for medical supervision. A particularly import-
ant advantage lies in the ability to adapt the materials, types of structure, and dimensions of
the needle to facilitate the delivery of macromolecules, nanoparticles, and vaccines [11,12].
(a) (b)
(c) (d)
FIGURE 18.2 Scanning electron micrographs of silicon microneedles. (a) Silicon microneedles micro-
fabricated using a modified form of the BOSCH deep reactive ion etching process. The microfabrication
process was accomplished at CCLRC Rutherford Appleton Laboratory (Chilton, Didcot, Oxon, UK).
The wafer was prepared at the Cardiff School of Engineering, Cardiff University, UK. Bar ¼ 100 mm;
(b–d) platinum-coated silicon microneedles prepared using a wet-etch microfabrication process per-
formed at the Tyndall National Institute, Cork, Ireland. Bar ¼ 1 mm (b), 100 mm (c,d).
silicon, a brittle material that regularly fractures on microneedle application, as the primary
structural component, microneedles have been fashioned from metal, polymer, and glass.
Metal microneedles were manufactured by electrodeposition of metal onto a polymer
or silicon micromold. Glass microneedles were created by conventional drawn-glass
micropipette techniques. Polymer microneedles were fabricated by melting polyglycolic
acid, polylactic acid, or polylactic-co-glycolic acid into polydimethylsiloxane (PDMS) micro-
molds [16] with these structures demonstrating up to three orders of magnitude increase in the
(a) (b)
FIGURE 18.3 Scanning electron micrographs of epidermal membrane treated with dry-etch and wet-
etch silicon microneedles. The epidermal membrane, consisting of stratum corneum and viable epider-
mis, was obtained by heat separation of full-thickness human breast skin. The tissue was immersed in
distilled water preheated to 608C for 60 s and the upper layers carefully peeled off from the dermal layer
using tweezers. Epidermal membranes were treated with microneedles for 30 s at an approximate
pressure of 2 kg=cm . (a) Dry-etch microneedle-treated epidermal membrane. Bar
2
¼ 200 mm; (b) wet-
etch microneedle-treated epidermal membrane. Bar ¼ 500 mm.
Elka Touitou / Enhancement in Drug Delivery 3203_C018 Final Proof page 342 3.10.2006 1:39pm
permeability of human cadaver skin to a low molecular weight trace molecule, calcein, and a
macromolecular protein, bovine serum albumin.
The ability to prepare microneedles from alternative material substrates confers important
advantages to the industrial exploitation of the technique. Whereas silicon microneedles are
generally fabricated in dedicated, and costly, clean room facilities, polymer and metal micro-
devices would be less expensive to mass produce as the raw materials are more available at a
reduced cost and the single-step molding techniques do not require specialist amenities. From
a clinical perspective many metals and polymers, as opposed to silicon, have established
biomaterial safety profiles and are more robust and less likely to shear upon skin application
and removal. A further advantage, specific to the use of polymeric material, relates to the
ability to manipulate the recipe to formulate biodegradable microneedles capable of either
in situ biological degradation following application or environmental degradation following
termination of use. Recently, potentially multifunctional polymeric microneedles comprising
a multilayer structure have also been prepared by Kuo and Chou [17].
A recent materials innovation in this field describes a method for coating microporous
calcium phosphate onto stainless steel acupuncture needles [18]. The incorporation of treha-
lose into the porous coating acted as a model fast-dissolving reservoir for the potential
loading and stabilizing of protein and DNA vaccines. The coated needles were shown to
remain intact following contravention of the stratum corneum and allow access of the coated
material to the viable epidermis. Importantly, no evidence of skin reaction, bleeding, or
infection was observed.
to passive diffusion of liquid into a microchannel created using a solid microneedle, and
provides the opportunity to pulse or continuously replenish the medicament over a period of
time. The extensive work of Zahn et al. [23] has shown that continuous pumping of fluid
through microneedles can be achieved for more than 6 h.
(a) (b)
FIGURE 18.4 Methylene blue staining of dry-etch (a) and wet-etch (b) microneedle-treated human skin.
Disruptions within the stratum corneum indicate microneedle penetration efficiency.
pDNA, i.e., pDNA formulated without additional complexing or targeting elements, through
microneedle-facilitated microchannels results in measurable levels of reporter gene expression
in excised human skin. Figure 18.5 shows typical results from skin transfection experiments.
En face imaging, following delivery of pCMVb reporter gene and staining with X-Gal, shows
microchannels stained positive for reporter gene expression (Figure 18.5a and Figure 18.5b).
Microchannel counterstaining with nuclear fast red shows that only a minority of micro-
channels were shown to be positive for gene expression (Figure 18.5a). These studies therefore
validate the skin explant organ culture conditions in maintaining the cellular viability of
excised skin and provide a realistic assessment of the current efficiency of the microneedle
technique for facilitating gene transfer. Photomicrographs showing the high level of reporter
gene expression in viable epidermal cells are presented in Figure 18.5c and Figure 18.5d.
Further studies are currently utilizing optimized microneedle devices to facilitate more
reproducible cutaneous gene delivery and explore additional factors that influence gene
expression in epidermal cells.
BD Technologies (Research Triangle Park, NC, USA) have used microfabricated silicon
microneedles, in their case termed microenhancer arrays (MEAs), to deliver genetic vaccines
to skin [39]. The application protocol involved applying a solution of DNA to the surface of
mouse skin and laterally scraping the microneedle array across the skin. The authors report a
2800-fold increase in reporter gene activity in comparison with conventional topical applica-
tion of controls and a more proficient and reproducible immune response in comparison with
needle injection. Encouragingly, lateral application of the MEA to human subjects confirmed
that the devices can breach the skin barrier with negligible to minimal skin irritation, no
damage to the microneedle array, and no reported incidence of infection.
(a) (b)
(c) (d)
FIGURE 18.5 Light photomicrographs of microneedle-treated human skin stained for b-galactosidase
expression. (a) En face stereomicroscopy of dry-etch microneedle-treated skin with nuclear fast red
counterstaining; (b) en face stereomicroscopy of wet-etch microneedle-treated skin; (c) hematoxylin and
eosin stained 12 mm cryosection of dry-etch microneedle-treated skin. Bar ¼ 100 mm; (d) hematoxylin
and eosin stained 12 mm cryosection of wet-etch microneedle-treated skin. Bar ¼ 100 mm.
Elka Touitou / Enhancement in Drug Delivery 3203_C018 Final Proof page 346 3.10.2006 1:39pm
A further advantage in utilizing the microneedle approach for vaccination and for the
delivery of proteins, peptides, and nucleic acids lies in the possibility of adjusting formulation
to meet stability and cost requirements. 3M Drug Delivery Systems (St Paul, MN, USA) and
ALZA Corporation (Mountain View, CA, USA) have developed methods to dry coat
microneedles with vaccine or drug to eliminate common pharmaceutical and resource con-
cerns over poor drug aqueous stability and cold-chain vaccine storage [11]. The future
application of these technologies is likely to have a significant future role to play in mass
immunization programs. ALZA have developed a microprojection patch (Macroflux) for the
controlled delivery of medicaments. Macroflux comprises a stainless steel or titanium micro-
projection array, fabricated from metallic foil, which is used to create superficial pathways in
skin and facilitate delivery of medicaments. This drug delivery system has been used to deliver
the peptide hormone desmopressin [40] and protein antigens [41] dry coated onto the projec-
tions, or in conjunction with iontophoresis for the administration of therapeutically relevant
quantities of oligonucleotides [42].
involves trajecting inert sharp particles, e.g., aluminum oxide crystals or other abrasive sub-
stances, onto the skin and subsequent removal of the crystals and abraded material. Using this
technique skin barrier function is transiently compromised with restoration within 24 h [53].
This method has been employed to enhance the topical permeation of vitamin C, 20-fold
increases in flux and skin deposition are reported when compared with intact skin [54], and
5-aminolaevulinic acid, up to 15-fold increase in skin permeation [48]. Interestingly in this latter
example, the permeation enhancement effect was significantly augmented by synergistic use of
electroporation or iontophoresis. O Herndon et al. [55] described the use of a gas-entrained
stream of 10–70 mm aluminum oxide particulates for the formation of microconduits for
transdermal delivery and sample acquisition [55]. The 50–200 mm depth channels formed by
microscission permitted rapid functional anesthesia following topical application of lidocaine
and allowed for the removal of blood glucose for monitoring purposes. An alternative method
for enhancing skin permeability uses standardized skin minierosion whereby an epidermal bleb
is created by suction to facilitate diffusion of analgesia for postoperative pain relief [56].
DNA-coated gold particles into target cells or tissues. The gold particles are typically around
1 mm in diameter and can penetrate through cell membranes, carrying the bound DNA,
typically 0.5–5 mg=mg gold, into the cell cytoplasm. Subsequent dissociation of the DNA
from the carrier particles allows the gene to be expressed [64]. Numerous studies have shown
the successful delivery and expression of genes using this method, with both reporter genes
and therapeutic plasmids having been transported to mammalian cells in culture [65],
oral mucosa [66], cornea [67], and animal skin [68–70]. Whereas expression of the transgene
is usually transient, lasting from a few days up to 4 weeks [71], Cheng et al. [68]
reported sustained luciferase activity in rat dermis one and a half years after in vivo particle
bombardment.
18.4 CONCLUSION
New technologies at the interface of engineering and biological sciences have provided the
drug delivery specialist with fresh opportunities for administering a range of therapeutics to
and through skin. The clinical exploitation of these technologies does not seem to be too
remote; however, there remain some limitations in these approaches, which necessitate further
investigation. It could be debated that creating channels, of any dimensions, in skin is
inherently dangerous and creates the opportunity for lasting skin damage and infection. Do
researchers in this area truly understand the kinetics and mechanisms of skin healing in
response to these assaults on skin integrity? Where a physical structure, such as a microneedle,
is used to penetrate the skin are any hyperproliferative, stress, or immune responses triggered?
As these devices are often designed to be used by patients in the absence of clinical interven-
tion is the hardware sufficiently simple to use in a safe and reproducible manner to create a
consistent number of channels at a reliable depth? As the stratum corneum acts as our
security blanket to infiltration by foreign bodies, are we wise in compromising it?
In my opinion these questions will be addressed, although it is clearly important that the
biological scientists work with the engineering scientists to ensure the development of these
technologies is treatment, disease, and patient focused rather than motivated by the tech-
nologies themselves.
ACKNOWLEDGMENTS
The significant contributions of Feriel Chabri, Marc Pearton, Sion Coulman, Ben Taunton,
Dr. Chris Allender, and Dr. Keith Brain are gratefully acknowledged. I am indebted to
Professor David Barrow, Tyrone Jones, and Kostas Bouris, Cardiff School of Engineering,
Cardiff University, and Dr. Anthony Morrissey and Nicolle Wilke, Tyndall National Institute,
Cork for their continued microfabrication support. I also thank Dr. Anthony Hann, Cardiff
School of Biosciences, for assistance with electron microscopy and Drs. Alexander Anstey,
Chris Gateley. and Helen Sweetland for clinical support.