rapidly – dissolvable microneedles for transdermal ... – dissolvable microneedles for...
Post on 29-Apr-2018
225 Views
Preview:
TRANSCRIPT
Rapidly – Dissolvable Microneedles for Transdermal Delivery via a Highly Reproducible Soft Lithography Approach
Katherine A. Moga1, Lissett R. Bickford1, Robert D. Geil1, Stuart S. Dunn1, Ashish A. Pandya1, Yapei Wang1, John H. Fain1,
Christine F. Archuleta1, Adrian T. O’Neill1, and Joseph M. DeSimone1,2,3
1The University of North Carolina at Chapel Hill, 131 South Rd, Chapel Hill, NC 27599, USA; 2North Carolina State
University, Raleigh, NC 27695 3Sloan-Kettering Institute for Cancer Research, New York, New York, 10021
kmoga@email.unc.edu
ABSTRACT SUMMARY Microneedle devices are an attractive method to
overcome the epidermis and effectively transport therapeutics transdermally. The fabrication of highly
reproducible polymer microneedles is described. These
completely dissolvable microneedle arrays are made on
flexible substrates using PRINT. Devices showed efficacy
in piercing the skin and delivering a fluorescent drug
surrogate to both ex vivo murine and human samples.
INTRODUCTION
Transdermal drug delivery is an attractive non-
traditional route of administration; it is non-invasive and
avoids first pass metabolism.1 One attractive method to
overcome the skin employs microneedle patches, arrays
of micron-sized projections for minimally-invasive drug delivery. Like hypodermic needles, these devices pierce
the skin but avoid nerve endings, causing no pain.1,2
Microneedles can transport therapeutics of virtually any
size through the skin, from small molecules to
nanoparticles.3 The low complexity of microneedle
devices may enable patient self-administration and
inexpensive fabrication. Thus, optimized microneedle
devices may offer the efficacy of a hypodermic needle with the benefits of transdermal delivery.
To overcome the barriers in fabrication of
microneedles seen previously, we have created
microneedle arrays using the Particle Replication In Non-
wetting Templates (PRINT®) technique.4 PRINT
combines a “top-down” method of soft lithography with
polymerization to create features on the nano- and micro-
scale with precise control of size, shape, and chemical composition. A wide range of materials including
polymers and pure drug could be used, and the mild
conditions required allow biologic cargo to maintain its
function throughout the process. PRINT can be adapted
on any scale, allowing affordable and quick fabrication.
Here, we demonstrate the fabrication of 100% water-
soluble microneedles on flexible substrates and their
ability to deliver a drug surrogate to ex vivo skin specimens. Array of discrete microneedles have been
manufactured via PRINT and collected on a flexible,
water-soluble substrates. This flexibility allows the array
of highly-dense microprojections to avoid this effect and
break the epidermis more efficiently. After application to
ex vivo murine and human skin specimins, the needle
patch remained long enough to allow the polymer to
dissolve and release a fluroescent drug surrogate. The substrate was then dissolved, leaving the entire
microneedle array (and drug payload) in the skin.
EXPERIMENTAL METHODS To fabricate PRINT microneedle patches, master
templates were first prepared using a tilted-rotated photolithography approach.5 First, a polished silicon
wafer was coated with thin an anti-reflective layer. A
thick layer of negative photoresist (SU-8) was
administered via spin coating and a mask (200 µm x 200
µm squares wtih 200 µm base-to-base spacing) was
applied. The complex was exposed to UV light at
incidence angles of 18-25°; the wafer was then rotated
90° about the surface normal for a total of four exposures. The resulting square pyramidal cavities were 360 µm
deep and had tip radii of curvature under 10 µm, seen via
Environmental Scanning Electron Microscopy (ESEM).
A positive replica of the master template was made
using polydimethylsiloxane (PDMS) as an intermediate.
A thick PDMS layer was cast upon the master,
centrifuged at 3000g, and cured overnight at 25°C. The
positive replica was then used to make PRINT-compatible molds from a photocurable perfluoropolyether (PFPE)
elastomer. A 0.2 wt% solution of 2,2-
diethoxyacetophenone in PFPE was cast onto the replica
and cured in a UV oven. The resulting molds are
consistent with the dimensions of the replicas,
reproducibly mimicking the SU-8 master templates.
Microneedles were fabricated using an adapted
PRINT process.4 Films of polyvinylpyrrolidone (PVP) were loaded with 0.1% rhodamine B fluorescent dye. A
film was mated to the PFPE mold and passed through a
heated nip at 105°C, filling the mold with discrete
microneedles. The filled mold was mated to a flexible,
water-soluble substrate (made from a blend of Plasdone, a
polyvinylpyrrolidone/polyvinylacetate blend, and triethyl
citrate) and passed through a heated nip at 65°C. The
mold was then removed, leaving a 100% water soluble microneedle patch. Microneedle morphology was
confirmed via ESEM and brightfield macroscopy.
Microneedle patches were tested on ex vivo nude
murine skin and human skin (obtained via the
Cooperative Human Tissue Network). Flexible PRINT
microneedle patches were “rolled” on with gentle force of
thumb and remained in the skin for a duration of either
10s or 10min. For the 10s tests, the patch backing was removed and the skin was exposed to green tissue-
marking dye for 5min. For the 10min tests, the patch
backing was then dissolved with <200 µL of tap water.
All skin samples were fixed for 2h in 2%
paraformaldehyde and left overnight in 15% sucrose in
1X PBS at 4°C. Control skin samples were also prepared;
these samples were not exposed to microneedles.
Tested murine and human skin samples were
embedded in Optimum Cutting Temperature medium and
cryosectioned. Sections (12 µm) were taken at -25°C.
Half of the sections imaged via fluorescent microscopy. The remaining sections were hematoxalin and eosin
(H&E) stained for brightfield microscopy imaging.
Staining was done using the procedure outlined by Cancer
Diagnostics for their CRYO-KIT prior to coverslipping.
RESULTS AND DISCUSSION Master templates, replicas, molds and PRINT
microneedles are shown in Figure 1A-D. The
microneedles retained the dimensions of the master with remarkable reproducibility. The flexibility of the array
can be seen in Figure 1E-F. The rigid microneedles
remained intact after the gentle bending of the array by
hand. Both the microneedles and the substrate were seen
to dissolve rapidly (~5min) in the presence of a few drops
of water. Therefore, novel 100% water-soluble
microneedle patches on flexible substrates can be made
quickly and reproducibly via PRINT processing.
Figure 1. ESEM images (A-D)of master template (A),
replica (B) and PFPE mold (C) and PVP microneedles
(D). Scale bar is 500 µm. Brightfield macroscopy (E-F) of microneedle arrays, showing flexibility. Scale bar is 1 cm.
The optimized PRINT microneedle arrays were first
tested in ex vivo murine skin samples (see Experimental
Methods). After H&E staining and brightfield imaging,
the control samples did not show any epidermal breach as
expected (Figure 2A); the skin was consistently smooth.
Evidence of epidermal breach was seen in skin sections
from both testing conditions (Figure 2B-C). Images of the unstained skin via fluorescent microscopy showed the
efficiency of the drug surrogate delivery. Seen in Figure
2D-F, a large qualitative difference in fluorescence
intensity was observed among the three samples. While
the control showed no fluorescence (Figure 2D), an
observable fluorescence was seen in the 10s test in
selective areas of the skin (Figure 2E). Comparatively,
considerably higher fluorescence intensity within the skin was seen for the 10min time period throughout the whole
skin section (Figure 2F). This confirms that the drug
surrogate was released from the needles and diffused
beneath the stratum corneum throughout the duration of
the patch application.
Figure 2. Images of ex vivo murine skin after testing with
PRINT microneedles. (A-C) Brightfield microscopic
images of skin sections after sectioning and H&E staining
[(A) control (B) 10s application (C) 10min application].
(D-F) Fluorescent microscopy images of skin after
sectioning. [(D) control (E) 10s application (F) 10min application]. Scale bar on all images is 35 µm.
In addition, pilot studies to determine the efficacy of
PRINT microneedles on human skin were also conducted.
Results from these experiments indicate that epidermal
breach and subsequent drug surrogate release. Figure 3A-
B shows a site of microneedle penetration and
corresponding rhodamine fluorescence in human skin.
While further optimization needs to be done, these findings support that PRINT microneedles may be used to
penetrate human skin and deliver loaded cargo.
Figure 3. Brightfield image (A) of a skin after
microneedle insertion for 10s. (B) Fluorescence image
after microneedle insertion for 10min. Scale bar is 70 µm.
CONCLUSION PRINT microneedle arrays (100% water-soluble)
were fabricated on flexible substrates. These arrays were
efficacious in piercing skin and delivering drug surrogate.
REFERENCES 1. Sullivan, S. P.; Murthy, N.; Prausnitz, M. R. Adv.
Mater. 2008, 20, 933-938.
2. Escobar-Chávez, J. J.; et al. J. Clin. Pharmacol.
2011, 51, 964-977. 3. Coulman, S. A.; et. al. Int. J. Pharm. 2009, 366, 190-
200.
4. Rolland, J. P.; et. al. J. Am. Chem. Soc. 2005, 127,
10096-10100.
5. Han, M.; Lee, W.; Lee, S. K.; Lee, S. S. Sensors and
Actuators A: Physical 2004, 111, 14-20.
ACKNOWLEDGMENTS Financial support was provided by the NIH Pioneer
Award, the University Cancer Research Fund at UNC
Chapel Hill, and the Charles N. Reilley Graduate
Fellowship through the UNC Department of Chemistry.
PRINT is a registered trademark of Liquidia
Technologies, Inc, and we thank them for their support.
top related