J. DRUG DEL. SCI. TECH., 23 (6) 577-581 2013
Recovery rate of rat skin micropores
made by dissolving microneedles
Y. Ito1*, N. Hamazaki1, H. Higashino1, N. Miyamoto1, Y. Murakami1, N. Inoue2, K. Takada1
Department of Pharmacokinetics, Kyoto Pharmaceutical University, Yamashina-ku, Kyoto, 607-8412, Japan
2
BioSerenTach Inc., Shimogyo-ku, Kyoto, Japan
*Correspondence: yukako@mb.kyoto-phu.ac.jp
1
The physiological recovery rate of skin was studied using dissolving microneedles (DMs) made of chondroitin sulfate. Chips of two kinds were
prepared having conical DMs as an array: (1) a 300 DM array chip having a sharp tip with mean diameter of 4.7 ± 0.2 μm, and a (2) 225 DM
array chip having a less-sharp tip with mean diameter of 15.3 ± 0.3 μm. Under anesthesia, DMs were inserted into abdominal rat skin from which
the hair had been removed. The kinetics of skin healing was studied by pathological and biochemical methods. At 5, 30, 45 min, and at 1 and 3 h
after the removal of the DM array chip, the skin was stained with 0.25 % Evans blue solution before pathologic observations were performed. The
skin physiology recovered to its normal state at 45 min with both DMs. Biochemical study was also performed by measuring the leaked amount
of lactose dehydrogenase (LDH) activity. The maximum leaks, 199.3 ± 13.0 % (225 DMs) and 153.4 ± 23.2 % (300 DMs) appeared at 5 min. In
both DMs, the micropores formed on the skin healed within 60 min. Pathological and biochemical examinations revealed that skin recovered to
the pre-treatment level within 1 h. The effect of the adhesion length of the chips onto the skin on the skin’s recovery rate was studied by attaching
two DMs to the rat skin for 6 h. Maximal leaks were detected at 5 min after administration, i.e., 157.8 ± 10.1 % for 300 DMs and 215.6 ± 5.4 %
for 225 DMs. However, the leakage returned to the pretreatment level at 60 min. Therefore, DMs were found to be a low-invasive TDDS for drugs.
Key words: Healing time - Dissolving microneedles (DMs) - Transdermal delivery - Skin - Rat.
molecules are absorbed. MN TDDSs are classified into four categories
[11]: (1) hollow-type MNs, which are extremely small needles through
which a drug solution is injected into the skin [12]; (2) coating-type
MNs, which are made of metallic and/or silastic substances onto which
a surface drug is coated [13]; (3) pierce-type MNs, which are made of
metallic or silastic microneedles that make microconduits in the skin,
after which a drug solution or cream is applied following removal of
the MNs [14]; and (4) dissolving microneedles (DMs), which are made
of soluble polymers such as sodium chondroitin sulfate and sodium
hyaluronic acid, where drug molecules are formulated into those base
polymer as a solid dispersion [15]. Among them, coated MNs are
the front-runner of MNs. Clinical phase II trials are underway in the
USA, where parathyroid hormone (PTH) is formulated for the treatment of osteoporosis [16]. On the other hand, we have been studying
two-layered DMs with water-soluble thread-forming biopolymers
such as chondroitin sulfate, dextran, hyaluronic acid, and albumin
used as the base. Chondroitin sulfate is a peptideglycan that exists in
biological tissues [17-22]. Chondroitin sulfate is used as an injection
preparation for the therapy of arthritis. Therefore, no safety problem
exists when chondroitin sulfate is used as the pharmaceutical additive
for dissolving microneedles. Drug molecules are formulated as a solid
dispersion with the base polymer. After the insertion of DMs into the
skin, the base polymer dissolves immediately. Then drug molecules
are released into the skin tissue, producing high availabilities of peptide/protein. For example, 98 % relative physiological availability of
insulin [23], 90.0-93.1 % bioavailability (BA) of desmopressin [24],
72.8-101.3 % BA of recombinant growth hormone [24], 65.9-69.0 %
BA of erythropoietin [25], and 79.9-117.8 % relative BA of interferon
[26] were obtained in our previous studies.
In those studies, 225-300 DMs were formed on a chip made
of cellulose acetate. To administer DMs into the skin, the chip was
pressed to the skin with collision speed of 0.8-2.2 m/s followed by
pressure of 7-22 N for 3 min when the polymer base dissolved within
the skin and drug molecules were released from DMs and absorbed
into systemic circulation. After the chip was removed from the skin,
The hurdle of developing new molecular entities as pharmaceuticals is becoming higher each year [1]. Therefore, R&D activity for life-expansion of active pharmaceutical ingredients (APIs)
has an important role in pharmaceutical industries. Most clinically
used dosage forms are oral and injection preparations. New dosage
forms, including drug delivery systems (DDSs) for oral and injection
preparations, are studied intensively. Although injection preparation
provides high bioavailability (BA) of drugs, e.g., BA is 100 % in the
case of IV injection preparation, injection preparation demands high
costs of both production and clinical treatment in clinics. In addition, patients, especially infants and child patients, often suffer from
pain. In contrast, oral preparations such as tablets and capsules are
convenient dosage forms, although drugs classified as class II and IV
compounds, as described by Amidon et al. [2], invariably suffer from
low-BA problems and high inter-subject variation of BA.
As an alternative to those preparations, percutaneous preparations
have attracted the interest of pharmaceutical scientists because they are
patient-friendly preparations. However, the percutaneous preparation
approved by the FDA as of 2007 amounted to only nine preparations
[3]. Because the barrier function of skin is very strong, only a limited
amount of a given drug is absorbed into systemic circulation. In fact,
human skin comprises three layers: the stratum corneum, epidermis,
and dermis. [4] The first is the 10-15-μm-thick outer layer, although
it is dead tissue. The stratum corneum has a strong primary barrier
function against drugs. The second barrier is the viable epidermis,
which has a 100-150 μm thickness and which contains tissues such as
living cells. However, no blood vessels exist in the epidermis [5]. To
increase the skin permeability of drugs, numerous approaches using
chemical enhancers, electric fields, ultrasound, and thermal methods
have been attempted [6-10]. However, the success of those transdermal
drug delivery systems (TDDSs) is limited because the skin has a strong
barrier function, which leads to low membrane permeability of drugs
through the skin. However, microneedles (MNs) present the possibility of cutting edge technology for the transdermal delivery of drugs.
MNs produce micropores on the skin stratum corneum into which drug
577
J. DRUG DEL. SCI. TECH., 23 (6) 577-581 2013
Recovery rate of rat skin micropores made by dissolving microneedles
Y. Ito, N. Hamazaki, H. Higashino, N. Miyamoto, Y. Murakami, N. Inoue, K. Takada
the micropores that were produced by DMs disappeared gradually
and the skin returned to its normal physiology. If the micropores do
not close for a long time, the possibility of the skin infection would
increase. Therefore, the kinetics of the skin healing, resealing, is an
important research project. To evaluate the skin damage in this study,
a pathological method using dye staining was used for the physical
assessment. In addition, a biochemical method was also used. Namely,
to evaluate skin irritation by cosmetics like shampoo and surfactants,
lactate dehydrogenase (LDH), leakage from skin tissue was measured
as a specific marker of cutaneous irritation [27-30]. Therefore, in this
report, the kinetics of skin healing was studied by both pathological
and biochemical approaches in rats.
the skin was recorded using a digital camera, (Nikon D-200; Nikon
Corp., Tokyo, Japan) under normal light. In the other groups of rats,
skin images were recorded at 30 min, 45 min, 1 h, and 3 h after application of the DM array chips.
In another group of rats, the LDH leakage was measured. At 5, 30,
45 min, and at 1 h and 3 h after the removal of DM array chip from
the abdominal rat skin, tissue paper, 2.0 × 2.0 cm, soaked with 10 μL
of phosphate buffered saline (PBS) was adhered to the skin for 1 min.
Then the leaked interstitial fluid was absorbed to tissue paper. LDH
was extracted from the tissue paper with 500 μL of PBS by shaking
for 5 min (recipro shaker SR-1; Taitec Corp., Saitama, Japan). After
the tube was centrifuged at 12,000 rpm for 15 min at 4 °C, the supernatant was obtained and used for LDH assay using a microplate reader
(MTP-300; Corona Electric Co. Ltd., Ibaraki, Japan) at an absorption
wavelength of 490 nm.
I. MATERIALS AND METHODS
1. Materials
Sodium chondroitin sulfate and cellulose acetate were obtained
from Wako Pure Chemical Industries Ltd. (Osaka, Japan). Evans
blue and brilliant blue (BB) were obtained from Nacalai Tesque Inc.
(Kyoto, Japan). Hydroxypropyl cellulose was obtained from Nippon
Soda Co. Ltd. (Tokyo, Japan). A lactate dehydrogenase (LDH) assay
kit was obtained (Cytotoxicity Detection Kit; Roche Diagnostics Corp.,
Manheim, Germany). Male Wistar-Hannover rats used in the study
and standard solid-meal commercial food were purchased (Japan SLC
Inc., Hamamatsu, Japan). All other materials were of reagent grade.
They were used as received.
6. Statistics
All values are expressed as their mean ± S.D. Statistical differences
were assumed to be significant when p < 0.05 (Student’s unpaired
t-test).
II. RESULTS
DM array chips of two kinds prepared in this study are presented
in Figure 1. Although the angle of their tip was the same (30°), the
sharpness of their tips differed. Table I shows the physical properties of the two DMs. The 300 DM array chip had 300 microneedles;
where the mean length was 491.9 ± 5.5 μm, the mean diameter of the
basement was 288.8 ± 5.2 μm and the mean diameter of the tip was
4.7 ± 0.2 μm. However, the 225 DM array chip had 225 microneedles
and a mean length of 489.6 ± 4.2 μm. The mean diameter of the basement was 290.6 ± 5.1 μm. The mean diameter of the tip was 15.3 ±
0.2 μm. No significant difference was found in the sizes of the two
2. Preparation of DM array chips
To 40 mg of sodium chondroitin sulfate, 0.5 mg of BB was added
if necessary, then 100 µl of 0.1 M phosphate buffered saline (PBS, pH
7.4) was added and chondroitin glue was obtained by mixing well.
After the glue was degassed under reduced pressure, it was dispensed
into a mold containing 225 or 300 inverted cone-shaped wells with an
area of 1.0 cm2. Each well had 500 µm depth and 300 µm diameter at
its top. The mold was covered with a 300 g steel plate. Then the glue
was filled into the wells and was dried at 40 °C for 2 h. A chip was
made of the mixture of cellulose acetate and hydroxypropyl cellulose
(10:1) using a tabletting machine, (Handtab-100; Ichihashi Seiki, Kyoto,
Japan). The chip, having a 2.0-mm thickness and 17-mm diameter,
was painted with the glue consisting of 15 mg of chondroitin sulfate
and 25 mL of distilled water by dipping into the glue solution. After
the plate was removed, the glue was painted over the chip and covered
on the mold. After drying under the pressure of a stainless steel plate
for 3 h, the chip was removed and DMs were obtained as arrays on a
chip.
3. Preparation of staining dye solution
Evans blue, 25 mg, was dissolved with 10 mL of saline.
4. Microscopic observation of DMs
A DM array chip of which the acral portion was stained with BB
was observed using a digital videomicroscope (VH-5500; Keyence
Co., Osaka, Japan) under normal light.
5. Recovery rate of micropores made on rat skin
Male Wistar-Hannover rats, 329 ± 21 g, were anesthetized with
sodium pentobarbital, 50 mg/kg. Body temperature was controlled
to 37 °C by warming during the experiment. Hair on the abdominal
region of each rat was removed using a shaver (ES7111; Panasonic
Inc., Osaka, Japan). The DM array chip was administered to the rat
skin using an applicator with collision speed of 2.0 m/s, followed by
secondary pressure of 2.5 N for 3 min. At 5 min after the DM array
chip was removed from the abdominal skin, soft tissue paper soaked
with 0.25 % Evans blue solution was attached to the skin for 30 s to
stain the micropores. After the soft tissue paper was removed, the skin
surface was wiped carefully with soft tissue paper. Then an image of
Figure 1 - Dissolving microneedle (DM) array chips of two kinds prepared for this study: (1) 300 DM array chip having a sharp tip and (2)
225 DM array chip having less sharpness. To clarify the shape of the
acral portion of DMs, brilliant blue was added to the base polymer.
578
Recovery rate of rat skin micropores made by dissolving microneedles
Y. Ito, N. Hamazaki, H. Higashino, N. Miyamoto, Y. Murakami, N. Inoue, K. Takada
J. DRUG DEL. SCI. TECH., 23 (6) 577-581 2013
Table I - Physical properties of the two dissolving microneedle array
chips (mean ± SD, n = 10).
Type
Number of
MN on a
chip
Length
(µm)
Diameter of the
basement
(µm)
Diameter
of the top
(µm)
300 DM
225 DM
300
225
491.9 ± 5.5
489.6 ± 4.2
288.8 ± 5.2
290.6 ± 5.1
4.7 ± 0.2
15.3 ± 0.3*
*p < 0.05 compared to type (1) MN.
DMs. However, 300 DMs had a sharper tip than 225 DMs. Figure 1
also shows an image of two types of DMs obtained using electron
microscopy. As shown in those photos, sharpness of the tip of 300 DM
was suggested.
Figure 2 portrays images of the stained micropores on the skin
formed by the insertion of DMs into the rat abdominal skin. At the
initial stage of this experiment, methylene blue was used to stain the
skin tissue. As methyl blue is a oxidation-reduction dye, it lost color
by oxidation-reduction enzymes that existed in the skin tissue. Instead,
Evans blue was used in this study, because it is nontoxic and used as
a diagnostic in the estimation of blood volume after its intravenous
injection; it is also used as a vital stain for following diffusion through
blood vessel walls. At 5 min after insertion, the micropores were detected clearly on the rat skin in both DMs. However, the micropores
stained with blue color became smaller as time passed. There was
not a great difference in the recovery rate between the two DMs. At
45 min after the administration, the stained micropores by both DMs
disappeared, which was ascribed to the recovery of the skin tissue
to its normal physiology. As a result, the skin of the two groups rats
healed within 60 min after insertion.
Figure 3 presents results of the LDH leakage from the rat skin
through micropores. At 5 min after the insertion of 225 DMs to the rat
skin, the leaked amount of LDH increased to twice the pretreatment
level. However, 300 DMs showed about 1.5-times higher LDH leakage than the pretreatment level. A significant difference was found
in LDH leakage between 225 DMs and 300 DMs. At 30 and 45 min,
LDH leakage amounts were about 151.5 ± 14.4 % and 124.5 ± 8.2 %
in 225 DMs. The 300 DMs also showed 122.0 ± 17.0 % and 113.8 ±
16.6 % LDH increases. Significant differences were found for LDH
leakage at 30 and 45 min between the two DMs. At 60 min after the
DM insertion, no significant difference in LDH leakage was found
as compared to the pretreatment levels. Therefore, skin physiology
reverted to pretreatment levels in both DMs.
To assess the effects of long-term adhesion of DMs to the skin,
DMs of both types were attached to the rat skin continuously for 6 h.
Thereafter, both pathological and biochemical studies were performed.
Figure 4 shows the time course of the recovery of stained micropores.
The micropores were stained with Evans blue until 45 min after the
insertion of DMs into rat skin. Thereafter, staining was not detected,
which showed the recovery of the skin tissue to the normal physiology. Figure 5 presents results elucidating LDH leakage after DMs
were attached to the skin for 6 h. The maximum leaks were detected
at 5 min after administration, 215.6 ± 5.4 % for 225 DMs and 157.8 ±
10.1 % for 300 DMs. The LDH leakage returned to the pretreatment
level at 60 min. Comparison to the results in Figure 3 reveals no
significant difference in the recovery rates of the skin between the
adhesive lengths of DMs to the skin.
Figure 2 - Time course of the image of the stained micropores made
on rat skin at 5 min after inserting 300 DM and 225 DM array chips. At
the predetermined time, the skin was stained with 2.5 % Evans blue
solution and was recorded using a digital camera.
Figure 3 - Time course of the leaked amount of LDH from the rat skin
through micropores made by 5 min adhesion of two kinds of DMs: black,
225 DM array chip; white, 300 DM array chip. Each point represents
the mean ± SD of 4-5 experiments. *Shows significant difference from
the control value (p < 0.05). **Shows significant difference from LDH
value of DM 300 (p < 0.05).
by which microconduits are made on the skin, after which a drug
solution or cream is applied. Because those microneedles are made
of metal or plastic, they belong to the category of medical devices.
When metal is used as the microneedle source material, microneedles
having a complex figure can be prepared. Prausnitz et al. prepared
arrow-shaped microneedles made of a stainless steel sheet and studied
the recovery rate of the micropores made on the skin by measuring
the skin impedance [31]. According to their results, microneedletreated skin sites had recovered barrier properties within 2 h, which
is a longer time than the results obtained in our study. To explain the
III. DISCUSSION
DMs are TDDS that offer particularly high safety because DMs
are made of water-soluble biopolymer such as sodium chondroitin
sulfate, dextran and hyaluronic acid. However, microneedles of three
kinds exist: (1) hollow microneedles connected to an infusion syringe,
(2) coated microneedles, and (3) metallic and/or silastic microneedles
579
J. DRUG DEL. SCI. TECH., 23 (6) 577-581 2013
Recovery rate of rat skin micropores made by dissolving microneedles
Y. Ito, N. Hamazaki, H. Higashino, N. Miyamoto, Y. Murakami, N. Inoue, K. Takada
The second reason is the shape of microneedles. Prausnitz et al. used
arrow-shaped stainless steel microneedles the surface of which was
treated by electropolishing [31]. However, the shape of our DMs was
conical, where the diameter of the tip portion was less and linearly
increased forward to the basement, the damage done to the skin was
thought to be less. The shape of our microneedle was conical, where
the theoretical height and diameter of the basement were 500 μm and
300 μm. We compared the recovery rate of the micropores produced
by two kinds of DM array chips having 225 and 300 DMs. The mean
diameter of 300 DMs was 4.7 ± 0.2 μm and that of 225 DMs was
15.3 ± 0.3 μm. The diameter of the tip of 225 DMs was about three
times greater than 300 DMs. When the tip portion of DM is inserted
into the skin, micropores are made physically. As the area of the tip
increased, the damage to the skin stratum corneum was thought to be
increased. As a result, the recovery rate of the micropores produced
by 300 DMs was faster than that made by 225 DMs. Those results
suggest the importance of the shape and size of microneedles on the
damage given to the inserted skin site. The third reason is the different
species examined in the safety study. Prausnitz used human volunteers.
In the present study, rats were used. Because the growth rate of the
body differs between humans and rats [32], the skin healing rates are
expected to differ between these species. Matsuo et al. studied skin
irritation caused by the insertion of DMs made of sodium hyarulonic acid [33]. They studied skin irritation by measuring the electric
impedance in rats. They reported that the electric impedance values
recovered within 2 h after the insertion. We also studied the effect of
the prolongation of DM adhesion to the rat skin on the recovery rate
of micropores on the skin. In our previous report, the systemically
absorbed amount of hydrophilic drug was dependent on the period
of adhesion of the DM array chip to the rat skin [34]. The absorbed
amount of sodium fluorescein (FL), which is a hydrophilic drug and
which is used as a diagnostic drug in ophthalmology, from the DM
array chip was increased from 0.18 ± 0.02 mg to 5.38 ± 1.99 mg by
increasing the adhesive period from 3 min to 6 h. At that time, the
damage of the rat skin was thought to be increased by the adhesion of
DM array chip for a long period. As a result, the absorbed amount of
FL was increased. However, our study based on pathology and biochemistry suggests that no significant difference exists in the recovery
rates of rat skin between 3 min adhesion and 6 h adhesion. Therefore,
we might state that the skin damage caused by DMs is tentative and
the recovery of the skin occurred within 1 h after the removal of DM
array chip.
Figure 4 - Time course of the image of the stained micropores made
on rat skin by 6 h adhesion of 300 DM and 225 DM array chips. At the
predetermined time, the skin was stained with 2.5 % Evans blue solution and was recorded using a digital camera.
*
In conclusion, the resealing time of skin surface micropores created by the collision of DMs was studied using both pathological and
biochemical methods. For a 300 DM array chip and 225 DM array chip
having the mean tip diameter of 4.7 ± 0.2 μm and 15.3 ± 0.3 μm, skin
physiology recovered to a normal state within 60 min. By increasing
the adhesion time from 3 min to 6 h, no significant difference was
found in the resealing time of the skin micropores. From those results,
DMs were found to be a low-invasive TDDS for drugs.
Figure 5 - Time course of the leaked amount of LDH from the rat skin
through micropores made by 6 h adhesion of two kinds of DMs: black,
225 DM array chip; white, 300 DM array chip. Each point represents
the mean ± SD of 4-6 experiments. *Denotes a significant difference
from the control value (p < 0.05). **Denotes a significant difference from
the LDH value of 300 DM (p < 0.05).
discrepancy in the results, we might state that three reasons are possible.
One is the difference of the material used to prepare microneedles.
In the earlier study, microneedles were prepared using a laser cutting
method and their surface cleaned by electropolishing. In contrast, the
sodium chondroitin sulfate used in our experiment is used clinically
as an injection preparation for arthritis therapy, where the clinical
dose is 20-300 mg. In other words, the material of DM is safety and
the amount of sodium chondroitin sulfate prepared in DM was too
far below the clinical dose. Therefore, the differences of material
for microneedles lead to the difference of the time for skin recovery.
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ACKNOWLEDGMENTS
This study was supported by a Ministry of Education, Culture, Sports,
Science and Technology (MEXT)-Supported Program for the Strategic
Research Foundation at Private Universities, 2008-2013. This study
was also supported by a grant-in-aid for scientific research provided
by MEXT, 2010-2013.
MANUSCRIPT
Received 8 February 2013, accepted for publication 7 August 2013.
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