Acta Biomaterialia xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Acta Biomaterialia
journal homepage: www.elsevier.com/locate/actabiomat

Poly-c-glutamic acid microneedles with a supporting structure design

as a potential tool for transdermal delivery of insulin
Mei-Chin Chen ⇑, Ming-Hung Ling, Setiawan Jati Kusuma
Department of Chemical Engineering, National Cheng Kung University, Tainan, Taiwan, ROC

a r t i c l e i n f o a b s t r a c t

Article history: Incomplete insertion is a common problem associated with polymer microneedles (MNs) that results in a
Received 12 February 2015 limited drug delivery efficiency and wastage of valuable medication. This paper presents a fully insertable
Received in revised form 18 May 2015 MN system that is composed of poly-c-glutamic acid (c-PGA) MNs and polyvinyl alcohol (PVA)/polyvinyl
Accepted 16 June 2015
pyrrolidone (PVP) supporting structures. The PVA/PVP supporting structures were designed to provide an
Available online xxxx
extended length for counteracting skin deformation during insertion and mechanical strength for fully
inserting the MNs into the skin. When inserted into the skin, both the supporting structures and MNs
Keywords:
can be dissolved in the skin within 4 min, thus quickly releasing the entire drug load from the MNs.
Bioavailability
Bolus release
To evaluate the feasibility and reproducibility of using the proposed system for treating diabetes, we
Microneedle administered insulin-loaded MNs to diabetic rats once daily for 2 days. The results indicated that the
Polypeptide hypoglycemic effect in the rats receiving insulin-loaded MNs was comparable to that observed in rats
Protein delivery receiving subcutaneous insulin injections. The relative pharmacological availability and relative bioavail-
Transdermal delivery ability of the insulin were in the range of 90–97%, indicating that the released insulin retained its phar-
macological activity. We observed no significant differences in the plasma insulin concentration profiles
between the first and second administrations, confirming the stability and accuracy of using the proposed
MN system for insulin delivery. These results indicated that the c-PGA MNs containing the supporting
structure design enable complete and efficient delivery of encapsulated bioactive molecules and have
great potential for the relatively rapid and convenient transdermal delivery of protein drugs.

Statement of Significance

Incomplete insertion of microneedles largely limits drug delivery efficiency and wastage of valuable med-
ication. To address this problem, we developed a fully insertable poly-glutamic acid microneedles with a
supporting structure design to ensure complete and efficient delivery of encapsulated drugs. The sup-
porting structures were designed to provide an extended length for counteracting skin compressive
deformation during puncture and mechanical strength for fully inserting the microneedles into the skin.
When inserted into the skin, both the supporting structures and microneedles can be dissolved in the skin
within 4 min, thus quickly releasing the entire drug load. This study demonstrated that the proposed
microneedle system featuring this unique design allows more convenient and efficient
self-administration of drugs into the skin.
Ó 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction require 2 or more injections of insulin daily, which is inconvenient

Type 1 diabetes is a chronic illness characterized by the body’s
inability to produce insulin because of the autoimmune destruc-
tion of pancreatic islet b cells. Children with type 1 diabetes melli-
tus (T1DM) have a lifetime dependence on exogenous insulin. Most
⇑ Corresponding author at: Department of Chemical Engineering, National Cheng
Kung University, Tainan 70101, Taiwan, ROC.
E-mail address: kokola@mail.ncku.edu.tw (M.-C. Chen).
and painful; moreover, it often leads to poor patient compliance
[1]. Many efforts have been devoted to explore more convenient
and palatable routes for insulin delivery, including intranasal,
intrapulmonary, oral, and transdermal administration [2–6].
Insulin can be absorbed across mucous membranes, whether in
the nose, lungs or intestine, but its bioavailability is very low.
Polymer-based particulate systems have also been widely investi-
gated as a carrier to increase insulin absorption by protecting insu-
lin from enzymatic degradation, increasing mucoadhesive ability,

http://dx.doi.org/10.1016/j.actbio.2015.06.021
1742-7061/Ó 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Please cite this article in press as: M.-C. Chen et al., Poly-c-glutamic acid microneedles with a supporting structure design as a potential tool for transder-
mal delivery of insulin, Acta Biomater. (2015), http://dx.doi.org/10.1016/j.actbio.2015.06.021
2 M.-C. Chen et al. / Acta Biomaterialia xxx (2015) xxx–xxx

and enhancing transport across the mucosa [4,5,7]. However, low
and erratic bioavailability remains major problems.
A recent study reported that compared with a subcutaneous
(SC) insulin administration, intradermal insulin delivery using a
hollow microneedle (MN) resulted in less insertion pain and led
to a faster onset and offset of insulin pharmacokinetics in children
with T1DM [8]. However, a custom-made holder and syringe pump
are required for controlling the retraction distance of hollow MNs
after insertion [9] and providing the infusion pressure to drive
insulin into the skin [10–12]. Moreover, the infusion pain associ-
ated with hollow MNs is not lower than that associated with SC
catheters [8].
Transdermal delivery of protein drugs by using dissolving poly-
mer MNs has attracted considerable attention recently [13–16].
Generally, therapeutic proteins are encapsulated within such poly-
mer MNs, and after insertion into the skin, the biocompatible poly-
mer dissolves within a few minutes to release the encapsulated
compounds. A syringe pump is not required for drug infusion, thus
preventing infusion pain and increasing compliance with insulin
therapy, particularly in needle phobic children.
Previous studies have demonstrated the feasibility of using
polymer MNs made of hyaluronic acid [12], a blend of starch and
gelatin [16], carboxymethyl cellulose [17], GantrezÒ AN-139 [18],
dextrin [19], and chondroitin sulfate [20] for transdermal insulin
delivery in vitro and in diabetic animals. However, inserting these
dissolving MNs fully into the skin is usually difficult because of the
wide needle geometry, low mechanical strength, and skin elasticity
[12,16,20,21]. The insertion depth is approximately one-third to
one-half of the MN length [12,16,20,21]. To achieve high drug
delivery efficiency, drugs were typically localized in the tip or
the acral portion of the MN [17,22,23], not in the entire needles.
Incomplete insertion of MNs largely limits drug loading capacity,
which may restrict the clinical applicability.
To address this problem, we developed a fully insertable
poly-c-glutamic acid (c-PGA) MN with a supporting structure
design to ensure complete and efficient delivery of encapsulated
insulin. The dissolvable supporting structure, made from polyvinyl
alcohol (PVA)/polyvinyl pyrrolidone (PVP), was connected to the
base of the c-PGA MN (Fig. 1). The supporting structure provides
mechanical strength and aids in overcoming skin deformation dur-
ing skin puncture, thus enabling full insertion of the MN into the
skin. Both the c-PGA MNs and the PVA/PVP supporting structures
can be dissolved in the skin’s interstitial fluid within 4 min, quickly
releasing the entire drug load. After release, no biohazardous
sharps or waste must be removed or discarded.
c-PGA is a high-molecular weight polypeptide comprising
c-linked glutamic acid units and a-carboxylate side chains pro-
duced by certain strains of Bacillus subtilis (formerly Bacillus natto)
[24]. It was used as the MN material because of its nontoxic,
biodegradable, and nonimmunogenic properties. A recent study
has reported that c-PGA has anti-inflammatory and
anti-angiogenic activities that was associated with significant
reduction in the expression of vascular endothelial growth
factor-A (VEGF-A) and its receptor (VEGFR-2) [25]. VEGF-A is
known to mediate pathologic angiogenesis in several inflammatory
disorders [26]. Furthermore, the end products from the degrada-
tion of this polypeptide are glutamates, common amino acids that
are nutritional to the skin. c-PGA-based nanoparticles have been
used as a carrier for delivering insulin, DNA, vaccines, and other
protein drugs [27–30]. However, an MN composed of pure c-PGA
exhibits a stability problem because of its high hygroscopic nature,
which may result in a reduction in the mechanical strength of the
MN, particularly in high-humidity environments. In this study,
pure c-PGA was blended with its hydrogel (i.e., c-polyglutamate
hydrogel; c-PGA hydrogel) to reduce hygroscopicity and produce
a more stable composite suitable for use as a needle material.
We investigated the effect of adding various concentrations of
c-PGA hydrogel into c-PGA on the hygroscopicity and skin inser-
tion ability of the prepared MNs. In vitro and in vivo transdermal
drug delivery properties of the MNs were evaluated after optimiz-
ing the composition. Moreover, the biological activity and
long-term storage stability of insulin encapsulated in the MNs
were also measured. To assess the efficacy and reproducibility of
using c-PGA MNs for transdermal insulin delivery, we injected
insulin-loaded MNs into streptozotocin (STZ)-induced diabetic rats
once daily for 2 days and compared the pharmacokinetics and
pharmacodynamics of MN-based insulin delivery with SC insulin
administration.

Fig. 1. Schematic illustrations of transdermal delivery of insulin using a fully insertable microneedle (MN) system, composed of poly-c-glutamic acid (c-PGA) MNs and
polyvinyl alcohol (PVA)/polyvinyl pyrrolidone (PVP) supporting structures. The supporting array can provide an extended length for counteracting skin deformation during
insertion and mechanical strength for fully inserting the MNs into the skin. After insertion, both the MNs and supporting structures can be rapidly dissolved in skin to release
encapsulated insulin.

Please cite this article in press as: M.-C. Chen et al., Poly-c-glutamic acid microneedles with a supporting structure design as a potential tool for transder-
mal delivery of insulin, Acta Biomater. (2015), http://dx.doi.org/10.1016/j.actbio.2015.06.021
 M.-C. Chen et al. / Acta Biomaterialia xxx (2015) xxx–xxx
2. Materials and methods
2.1. Materials
c-PGA (Na+ form, MW approximately 1000 kDa) and c-PGA
hydrogel powder (Na+ form, water absorption capability =
1654 g water/g hydrogel) were purchased from Vedan Enterprise
Corp (Taichung, Taiwan). Fluorescein 5(6)-isothiocyanate (FITC;
MW = 389 Da), blue-dextran (MW = 2000 kDa), STZ, PVA
(MW = 6000), PVP (MW = 10,000), phosphate buffered saline
(PBS) and insulin (from bovine pancreas, P25 units/mg, MW
approximately 5.8 kDa) were purchased from Sigma–Aldrich
(St. Louis, MO, USA). Insulin ELISA kit, and Glucose CII-Test kit were
purchased from Mercodia AB (Uppsala, Sweden) and Wako Pure
Chemical Industries (Osaka, Japan), respectively. Polydimethylsilo
xane (PDMS; Sylgard 184), blue tissue-marking dye, and the
Optimum Cutting Temperature (OCT) compound were purchased
from Dow Corning (Midland, MI, USA), Shandon (Kalamazoo, MI)
and Tissue-Tek (Sakura Finetek, Torrance, CA, USA), respectively.
2.2. Fabrication of drug-loaded microneedles
A MN master structure was created using an electro-discharge
machining process (Micropoint Technologies Pte, Ltd., Singapore).
MN molds were made from PDMS to inverse-replicate the master
structure, following a published procedure [16]. The obtained
PDMS molds were repeatedly used to make polymer MNs.
A 2-step casting process was used to fabricate drug-loaded MN
patches. A c-PGA gel solution (30 wt.%) containing drug was used
to fill the first layer for the MN part, and a PVA/PVP solution
(50 wt.%) was used to fill the second layer for the supporting struc-
ture part. The c-PGA gel solutions containing 0%, 25%, 50%, and 75%
(w/w) hydrogels were prepared by dissolving c-PGA and hydrogel
powder at various blending ratios (100:0, 75:25, 50:50, and 25:75)
in deionized (DI) water and stirred overnight. The PVA/PVP solu-
tion was prepared by dissolving 5 g of PVA and 5 g of PVP into
10 ml of DI water.
To prepare the insulin-loaded solution, two milligrams of insulin
or FITC-insulin dissolved in 0.3 ml of 0.1 M HCl solution was added to
the c-PGA gel solution (10 ml, c-PGA/hydrogel weight ratio of 50:50)
and mixed well (final pH of approximately 6.0). Approximately 1 g of
the drug-loaded c-PGA solution was applied to the PDMS mold as
the first layer and centrifuged in a swinging bucket rotor (221.12
V03, Hermle Labortechnik GmbH, Wehingen, Germany) at
5100 rpm (3880g) at 30 °C for up to 2 h to fill the MN mold cavities.
Gel remaining on the mold surface was removed and saved for future
use. The filled mold was centrifuged again for 1 h without sealing
caps and then air dried at room temperature overnight.
The second layer of PVA/PVP solution (approximately 250 mg)
was subsequently placed on the dried first layer, followed by fur-
ther centrifugation at 5100 rpm and 30 °C for 30 min. The filled
mold was air dried at room temperature overnight and then put
in an oven at 37 °C for 1 day. Finally, the drug-loaded MN patch
was gently peeled from the mold and examined using a stereomi-
croscope (SZ-61, Olympus, Olympus Corporation, Tokyo, Japan).
For the blue-dextran-loaded samples, blue-dextran (0.1 g) can be
directly added and mixed with the c-PGA gel solution (10 ml). The
same casting process was also used for the blue-dextran-loaded
MNs.
2.3. Synthesis of FITC-insulin
To visualize drug diffusion in the skin, insulin was fluorescently
labeled with FITC using the method described in the literature [31].
Please cite this article in press as: M.-C. Chen et al., Poly-c-glutamic acid microneedles with a supporting structure design as a potential tool for transder-
mal delivery of insulin, Acta Biomater. (2015), http://dx.doi.org/10.1016/j.actbio.2015.06.021
3
2.4. Hygroscopicity test
MNs were first placed in a dry desiccator for two days to allow
all samples to have equivalent conditions before moisture absorp-
tion measurement. Then the samples were stored in a humidity
chamber at a relative humidity (RH) of 55%. Subsequently, MNs
were removed at pre-determined intervals, and the weight of
MNs was measured. The moisture uptake by the MNs was calcu-
lated as the percent increase in MN weight [12].
2.5. In vitro skin insertion
To evaluate the skin insertion capability, MNs were inserted
into porcine cadaver skin for 4 min using a homemade applicator.
The MN insertion site on the skin surface was exposed to blue
tissue-marking dye for 1 min to identify the stratum corneum per-
foration sites and calculate the insertion ratio. Histological sections
of the skin were then excised to determine the insertion depth. The
insertion ratio for each test sample was calculated by dividing the
number of blue spots on the skin after insertion by the number of
array needles. Insertion depth was defined as the average penetra-
tion of needles successfully inserted into the skin from 5 MN
patches.
To prepare histological specimens, MN insertion sites were
excised from the skin and embedded in an OCT compound for
cryosection. The frozen OCT skin samples were then sliced into
5-lm-thick sections using a cryotome (Shandon Cryotome E,
Thermo Electron Corporation, USA) and then analyzed using an
inverted fluorescence microscope (IX-71, Olympus, Tokyo, Japan).
2.6. In vitro release of insulin from microneedles
Insulin-loaded MNs were inserted into pig cadaver skins by
using the applicator and secured to the skin using dermal tape.
At a specified time interval, the patches were removed from the
skin, and the insertion site was then tape-stripped 10 times with
3 M Transpore™ tapes to remove the residual insulin on the skin
surface. The amount of insulin delivered into the skin was calcu-
lated by subtracting the amount of insulin remaining in the MNs
after insertion and remaining on the skin surface from the amount
originally encapsulated in the MNs.
To determine the loading and residual amounts of insulin in
MNs, the patches before and after skin insertion were separately
dissolved in phosphate buffered saline (PBS) with mixing at 4 °C
for 1 day to release the encapsulated insulin. The stripped tapes
were also soaked in PBS for 1 day at 4 °C to recover the insulin
on the tape. The amount of insulin extracted from the MNs and
the stripped tape was determined using high-performance liquid
chromatography (HPLC) according to our previous work [16].
2.7. Biological activity and storage stability of insulin encapsulated in
microneedles
Biological activity of the encapsulated insulin was measured
using an insulin-ELISA kit following a standard protocol [32]. To
extract insulin from the MNs, insulin-loaded MN patches were dis-
solved in PBS with mixing at 4 °C for 1 day. Insulin content was
analyzed for (A) untreated insulin, insulin encapsulated in MNs
dissolved after (B) 1 day and (C) 1 month of storage at 37 °C, and
(D) insulin thermally treated at 80 °C for 1 h.
To determine the storage stability of insulin encapsulated in
c-PGA MNs, insulin-loaded MNs were stored at 20, 4, 25, and
37 °C for 1 month. The encapsulated insulin was then extracted
from the MNs and the insulin concentration was measured from
the extracted buffer by HPLC.
4 M.-C. Chen et al. / Acta Biomaterialia xxx (2015) xxx–xxx
2.8. Animal study
The Institutional Animal Care and Use Committee of National
Cheng Kung University approved all animal protocols, and experi-
ments were conducted according to the guidelines of the
Laboratory Animal Center of National Cheng Kung University.
Four-week-old male Sprague Dawley (SD) rats weighing
200 ± 25 g were used. The rats were anesthetized with an intra-
muscular injection of Zoletil 50 (35 mg/kg) and Rompun (2 mg/kg),
and their back hair was removed using an electric shaver. The
application site on the bare rat skin was gently swabbed with
70% ethanol and allowed to dry [33].
2.8.1. Transepidermal water loss (TEWL) measurement
MNs were administered to the rat dorsal skin using the applica-
tor and removed from the skin after administration for 4 min.
Negative control rats received hair trimming only. After treatment,
TEWL was measured using a Delfin VapoMeter (Delfin
Technologies Ltd., Kuopio, Finland) according to the manufac-
turer’s instructions. At least three readings were taken at every
time point. TEWL readings were also recorded at 0, 10 and
30 min, 1, 2, 3, 4, 6, and 9 h. The experiment was repeated using
five rats per group.
2.8.2. In vivo transdermal delivery of insulin using MNs
To visualize the penetration of the FITC-insulin within the rat
skin, the MN treated skin was excised and observed using a confo-
cal laser scanning microscope (CLSM; FluoView FV1000, Olympus
Corporation, Tokyo, Japan) at an excitation wavelength of 488 nm
[34]. Images were obtained in the xy-plane. The initially scanned
skin surface (z = 0 lm) was defined as the imaging plane of the
brightest fluorescence with a morphologic characteristic of the
stratum corneum surface. The 3D confocal reconstruction images
were obtained using the xyz-stack [35].
To visualize cutaneous permeation of insulin in living rats, the
live anaesthetized rats were imaged at 10 min and 1 h after treat-
ment with MNs loaded with or without FITC-insulin using an In
Vivo Imaging System (IVIS; Xenogen 200, Caliper Life Sciences,
Alameda, CA). This IVIS system was used to collect and analyze
all fluorescence data, which are expressed as photon-flux
(photons/s/cm2/steradian) [36].
2.8.3. Transdermal delivery of insulin to diabetic rats
Rats were intraperitoneally injected with 50 mg/kg of strepto-
zotocin dissolved in a citric acid buffer solution (pH 4.5) to produce
a diabetic animal model. Animals with blood glucose levels
exceeding 300 mg/dL determined using a One Touch glucometer
(LifeScan, Inc., Milpitas, CA, USA) were labeled diabetic [12]. Prior
to the experiment, diabetic rats fasted for 12 h but received water
ad libitum. All animals were anesthetized and shaved as described.
The following groups of diabetic rats (n = 5 for each group) were
studied after drug administration once daily for 2 days: (1) the
control group, where unloaded MNs were applied to the back skin
and fixed with tape; (2) the SC group, where an insulin solution
(0.2 IU) was subcutaneously injected into the abdominal skin using
a hypodermic needle, and (3) the insulin-loaded MN group, where
insulin-loaded MNs (0.2 IU/patch) were applied to the back skin
and fixed with tape.
In all groups, blood samples were collected from the jugular
vein for 30 h and then centrifuged at 3000 rpm for 5 min to imme-
diately separate the plasma. Plasma glucose levels were deter-
mined using a glucose CII-test kit, and the initial levels at 0 h
were considered to be 100%. The percentage change in plasma glu-
cose levels at each time interval after dosing was calculated based
on these initial values. Plasma insulin concentrations were mea-
sured using an insulin-ELISA kit [12].
Please cite this article in press as: M.-C. Chen et al., Poly-c-glutamic acid microneedles with a supporting structure design as a potential tool for transder-
mal delivery of insulin, Acta Biomater. (2015), http://dx.doi.org/10.1016/j.actbio.2015.06.021
2.8.4. Pharmacodynamic and pharmacokinetic analyses of insulin after
application of insulin-loaded microneedles
The maximum plasma insulin concentration (Cmax) and the time
point of maximum plasma insulin concentration (Tmax) were deter-
mined by plotting the plasma insulin concentration (lIU/ml) over
time. The relative bioavailability (RBA) was calculated using the
following equation:
RBA ð%Þ ¼ ðAUCMN  DoseSC Þ=ðAUCSC  DoseMN Þ  100
where AUCMN shows the area under the curves after applying the
insulin-loaded MNs, and AUCSC indicates the area under the curves
after the SC injection of insulin [12].
The minimum glucose level (Cmin) and the time point of mini-
mum glucose level (Tmin) were determined by plotting the percent-
age change from the initial levels of plasma glucose over time. The
relative pharmacological availability (RPA) was calculated using
the following equation:
RPA ð%Þ ¼ ðAACMN  DoseSC Þ=ðAACSC  DoseMN Þ  100
where AACMN shows the area above the curves after applying the
insulin-loaded MNs, and AACSC indicates the area above the curves
after the SC injection of insulin.
2.9. Statistical analysis
For this study, we compared the 2 groups using the one-tailed
Student t test by employing statistical software (SPSS, Chicago,
Ill, USA). This study presents data as the mean ± SD. A difference
of p < 0.05 was considered statistically significant.
3. Results
3.1. Characterization of microneedles
We fabricated c-PGA MNs equipped with PVA/PVP supporting
structures by using a simple 2-step casting process. The patch com-
prised 81 (9  9) needles. The base width and height of both the
MN and supporting structure were 300 and 600 lm, respectively
(Fig. 1). To visualize the drug distribution within the MN,
blue-dextran was used as a model drug. As shown in Fig. 2, the
drug (blue) was uniformly encapsulated in the entire MN.
c-PGA, a hydrophilic polypeptide, has been used in cosmetics as
a moisturizer. However, because of high water-solubility, main-
taining the stiffness and sharpness of c-PGA MNs is difficult
because they easily absorb moisture from the atmosphere. In this
study, we incorporated c-PGA hydrogels into the MN formulation
to reduce hygroscopicity and improve stability. The c-PGA hydro-
gel, prepared by crosslinking c-PGA by using polyglycerol polygly-
cidyl ether, was obtained from Vedan Enterprise Corp [37]. We
hypothesized that incorporating these covalently cross-linked
hydrogels can ensure resistance to MN deformation during mois-
ture absorption.
To evaluate the hygroscopicity of MNs containing various ratios
of c-PGA hydrogel, the weight of the MNs was measured over a
24-h period at a RH of 55%. The water uptake of all samples
increased rapidly during the first 3 h and gradually reached satura-
tion at 24 h (Fig 3). Water absorption at saturation decreased sub-
stantially with an increase in hydrogel content. Fig. 2 shows the
morphology of blue-dextran-loaded MNs during moisture uptake.
After exposure to the RH 55% environment for 3 h, the MNs con-
taining 0 and 25 wt.% hydrogel gradually deformed and became
blunt; however, the samples containing 50 and 75 wt.% hydrogel
maintained their shape and a sharp tip.
To assess the skin insertion capability of the MNs after moisture
absorption, we inserted samples into porcine cadaver skin. After
 M.-C. Chen et al. / Acta Biomaterialia xxx (2015) xxx–xxx 5

Fig. 2. Hygroscopicity of blue-dextran-loaded microneedles (MNs) containing 0, 25, 50, and 75 wt.% hydrogel. Bright-field micrographs of MNs after exposure to a relative
humidity environment of 55% for 0, 1, and 3 h.

skin insertion capability (Fig. 4) between the samples containing

50 and 75 wt.% hydrogel. The MN containing 50 wt.% hydrogel
:HLJKW RI 01V  LQFUHDVH RI LQLWLDO

was used for the subsequent studies because of its ease of handling
and preparation.

3.2. In vitro drug release profile of insulin-loaded microneedles

To evaluate the insulin release from the MNs, we inserted

insulin-loaded MNs into porcine cadaver skin and monitored the
drug release. All needles completely dissolved in the skin and
rapidly released the encapsulated insulin within 4 min after con-
tact with the skin’s interstitial fluid (Fig. 5).

Q  3.3. In vivo transdermal delivery of FITC-insulin from the microneedles

To evaluate the feasibility of the transdermal delivery of insulin

7LPH K by using c-PGA MNs, we inserted FITC-insulin-loaded MNs into the
Fig. 3. Hygroscopic weight gain of microneedles containing 0, 25, 50, and 75 wt.%
hydrogel after exposure to a relative humidity environment of 55%.
insertion, the test skin was stained with a blue tissue marking dye
to calculate the insertion ratio. The skin surface of all tested groups
exhibited a complete array of blue spots (9  9) corresponding to
the MN insertion sites (Fig. 4), indicating that the insertion ratio
of all groups was 100%. According to the histological sections, the
insertion depths of the MNs containing 0, 25, 50, and 75 wt.%
hydrogel were 510 ± 50, 511 ± 37, 667 ± 27, and 658 ± 26 lm,
respectively, at 1 h and 415 ± 85, 418 ± 39, 647 ± 21, and
650 ± 15 lm, respectively, at 3 h (n = 5 for each group). Only the
MN samples containing 50 and 75 wt.% hydrogel retained mechan-
ical strength and could be completely inserted (>600 lm), even
after exposure to the 55% RH environment for 3 h (Fig. 4). These
results indicated that adding hydrogel (at a ratio of P50 wt.%) to
the MNs improved the MN resistance to moisture. We observed
no significant difference in the moisture absorption (Fig. 3) and
skin on the back of SD rats. After insertion for 4 min, the MNs cre-
ated micrometer-sized cavities in the skin and released the encap-
sulated FITC-insulin (green) at the puncture sites (Fig. 6a and a1).
The arrows in Figs. 6a indicate the skin puncture sites created by
the MNs. To visualize the penetration of FITC-insulin in the vertical
direction of the rat skin, the MN puncture sites were imaged and
recorded at increasing depths from the skin surface by using a
CLSM. The diffusion depth of FITC-insulin was approximately
800 lm at 4 min post-administration (Fig. 6b and b1), indicating
that insulin can be directly delivered to the dermis layer, which
is beneficial for drug absorption into the systemic circulation.
These results reveal that the c-PGA MNs have the ideal strength
for penetrating living skin and can then rapidly dissolve to release
encapsulated drugs.
To characterize the cutaneous permeation of the released insu-
lin, we inserted unloaded (negative control) or FITC-insulin-loaded
MNs into rat skin for 4 min and then removed the patches from the
skin. Rats were visualized at 10 min and 1 h after MN delivery by

Please cite this article in press as: M.-C. Chen et al., Poly-c-glutamic acid microneedles with a supporting structure design as a potential tool for transder-
mal delivery of insulin, Acta Biomater. (2015), http://dx.doi.org/10.1016/j.actbio.2015.06.021
6 M.-C. Chen et al. / Acta Biomaterialia xxx (2015) xxx–xxx

3RUFLQH VNLQ +LVWRORJLFDO 6HFWLRQ

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Fig. 4. In vitro skin insertion capability of microneedles (MNs) containing 0, 25, 50, and 75 wt.% hydrogel after exposure to a relative humidity environment of 55% for 3 h. The
left column shows the bright-field micrographs of porcine cadaver skin after MN insertion and staining with blue tissue marking dye. The right column shows the
corresponding histological section of MN puncture sites.

a b
Cumulative release of insulin (%)

0 s 2 min
(n = 5)

30 s 3 min

1 min 4 min

Insertion time (min)

Fig. 5. In vitro insulin release from microneedles (MNs). (a) MN dissolution before (0 s) and after insertion into porcine cadaver skin for 30 s, 1, 2, 3, and 4 min. (b) In vitro
drug release profile. The loading amount of insulin was 0.20 ± 0.02 IU per patch (n = 5).

using the IVIS. The sites that received FITC-insulin-loaded MNs
exhibited a strong fluorescent signal, and the fluorescent intensity
and area decreased noticeably 1 h after application (Fig. 7), indicat-
ing that the FITC-insulin released from the MNs gradually diffused
into the skin.
3.4. Recovery of skin barrier function after microneedle insertion
We used TEWL measurement to evaluate the skin barrier func-
tion after MN treatment. The TEWL value in the treated skin area
increased immediately after MN insertion (Fig. 8a). However, this
value gradually reverted to a level similar to that of intact skin
(control group) within 6 h after removing the MNs, indicating that
the skin barrier function was recovered, and that the skin damage
induced by the MNs was reversible. Monitoring the presence of
skin punctures also provided a preliminary indication of skin
resealing. Fig. 8b shows the micrographs of puncture marks on
the skin at 0 min, 1 h, and 6 h after MN application. As shown, evi-
dence of microchannel repair and resealing was apparent 6 h
post-application. Previous studies have reported that c-PGA can

Please cite this article in press as: M.-C. Chen et al., Poly-c-glutamic acid microneedles with a supporting structure design as a potential tool for transder-
mal delivery of insulin, Acta Biomater. (2015), http://dx.doi.org/10.1016/j.actbio.2015.06.021
 M.-C. Chen et al. / Acta Biomaterialia xxx (2015) xxx–xxx 7

a
a
b
b

Z 0 µm 300 µm Z 200 µm 300 µm Z 300 µm 300 µm

a1

Z 400 µm 300 µm Z 500 µm 300 µm Z 600 µm 300 µm

b1
b 1
Z
800 µm

3D reconstruction image Z 700 µm 300 µm Z 800 µm 300 µm Z 900 µm 300 µm

Fig. 6. In vivo transdermal delivery of insulin from microneedles (MNs). (a) Fluorescence and (a1) merged images of bright-field and fluorescence of histological sections of
skin puncture sites (arrows). (b) Confocal micrographs and (b1) their 3D reconstruction images of penetration of FITC-insulin (green) across the skin at varying depths after
insertion for 4 min. The green fluorescence indicates the released FITC-insulin.

Fig. 7. In vivo fluorescence images of Sprague Dawley (SD) rats at 10 min and 1 h after treatment of unloaded and FITC-insulin-loaded microneedles (MNs).

accelerate wound healing because of its antiinflammatory property
and ability to promote cell migration and proliferation [38]. The
results indicated that using this system for drug delivery is harm-
less and the rapid skin recovery may reduce the risk of wound
infection.
3.5. Biological activity and storage stability of insulin encapsulated in
microneedles
To confirm that the MN fabrication and encapsulation processes
do not lead to the denaturation of encapsulated biomolecules, we

Please cite this article in press as: M.-C. Chen et al., Poly-c-glutamic acid microneedles with a supporting structure design as a potential tool for transder-
mal delivery of insulin, Acta Biomater. (2015), http://dx.doi.org/10.1016/j.actbio.2015.06.021
8 M.-C. Chen et al. / Acta Biomaterialia xxx (2015) xxx–xxx

a b
0h

(n = 5)
TEWL (g/m2h)
1h

6h

Time (h)
Fig. 8. Skin resealing and recovery after microneedle (MN) treatment. (a) Transepidermal water loss (TEWL) from the skin at different time points after MN application (n = 5
for each group). (b) Skin puncture marks at 0, 1 and 6 h post-treatment.

Table 1
Stability of insulin-loaded microneedles (MNs) after storage at 20, 4, 25, and 37 °C
Q 
100 for one month (n = 6).

Temperature (°C) Insulin remaining in MNs (% of initial)

%LRORJLFDO DFWLYLW\ 
80
60
40
20
0 Fig. 10a shows the plasma insulin concentrations in diabetic
A B C D
,QVXOLQ WUHDWPHQW
Fig. 9. Biological activity of insulin encapsulated in microneedles (MNs): (A)
untreated insulin, insulin encapsulated in MNs that were dissolved after (B) 1 day
or (C) 1 month of storage at 37 °C, and (D) insulin thermally treated at 80 °C for 1 h
(n = 5 for each group).
examined the biological activity of insulin before and after encap-
sulation in MNs by using an insulin-ELISA kit [16,39]. Fig. 9 shows
the biological activity of untreated insulin, insulin encapsulated in
MNs and subsequently released after storage at 37 °C for 1 day or
1 month, and insulin thermally treated at 80 °C for 1 h (negative
control). The activity of insulin after MN encapsulation and storage
at 37 °C for 1 day was statistically indistinguishable (p > 0.05, n = 5)
from that of the untreated insulin solution. Furthermore, after stor-
age at 37 °C for 1 month, the insulin activity in the MN group
decreased only slightly.
Table 1 shows the storage stability of insulin-loaded MNs after
storage at 20, 4, 25, and 37 °C for 1 month. More than 90% of the
encapsulated insulin remained in the MNs in all groups. These
results indicated that encapsulating insulin within the c-PGA
MNs did not significantly reduce the biological activity; further-
more, these MNs were stable for at least 1 month at 25 and
37 °C, a characteristic that may reduce the requirement for cold
chain storage and transportation.
Please cite this article in press as: M.-C. Chen et al., Poly-c-glutamic acid microneedles with a supporting structure design as a potential tool for transder-
mal delivery of insulin, Acta Biomater. (2015), http://dx.doi.org/10.1016/j.actbio.2015.06.021
20 98.9 ± 0.9
4 97.8 ± 0.3
25 92.6 ± 1.6
37 91.0 ± 1.8
3.6. Transdermal delivery of insulin to diabetic rats
rats after SC insulin injection and application of unloaded (control)
MNs and insulin-loaded MNs once daily for 2 days. The plasma
insulin concentration increased rapidly and reached a maximal
value at 1 h and then returned to normal 6 h after the application
of the insulin-loaded MN. We observed no significant difference in
the plasma insulin concentration profile between the MN group
and the SC group after the first (at 0 h) and second (at 24 h) treat-
ments; thus, the stability and accuracy of the insulin delivered
using the c-PGA MNs was confirmed.
Fig. 10b shows the corresponding plasma glucose levels. In the
first administration, the plasma glucose level was reduced to 46%
of the initial value 1 h after treatment with the insulin-loaded
MNs. Furthermore, the hypoglycemic effect was comparable to
that obtained in the SC group treated with the same dose of insulin
(0.2 IU). After the second administration at 24 h, a similar plasma
glucose level profile was observed in rats treated with
insulin-loaded MNs, confirming the reproducibility of using
c-PGA MNs for insulin delivery. These results indicated that the
proposed c-PGA MNs are as effective as currently used syringes
for T1DM patients.
Tables 2 and 3 show the pharmacokinetic parameters for
plasma insulin concentrations and pharmacodynamic parameters
of the plasma glucose levels, respectively. The relative bioavailabil-
ity (RBA) and pharmacological availability (RPA) values of the
insulin-loaded MN group were in the range of 90–97%. These
results indicated that the pharmacological activity of insulin was
maintained after release from the c-PGA MNs and that the released
 M.-C. Chen et al. / Acta Biomaterialia xxx (2015) xxx–xxx 9

a b

3ODVPD LQVXOLQ FRQFHQWUDWLRQ µ,8PO

Q 

3ODVPD JOXFRVH OHYHO  RI LQLWLDO

Q 

7LPH K 7LPH K

Fig. 10. Transdermal delivery of insulin to diabetic rats. (a) Plasma insulin concentrations vs. time profiles and (b) plasma glucose levels vs. time profiles of diabetic rats after
administration of unloaded (control) and insulin-loaded microneedles (MNs) and subcutaneous injection of insulin (insulin SC) (n = 4 for each group).

Table 2
Pharmacokinetic parameters for plasma insulin concentrations of diabetic rats after administration of unloaded microneedles (MNs), insulin-loaded MNs (0.2 IU), and a
subcutaneous injection of insulin (insulin SC, 0.2 IU) (n = 4).

Group C1max (lIU/ml) C2max (lIU/ml) T1max (h) T2max (h) AUC0?6 (lIUh/ml) AUC24?30 (lIUh/ml) RBA1 (%) RBA2 (%)
Unloaded MNs 0 0 – – 0 0 0 0
Insulin-loaded MNs 203 ± 22 205 ± 20 1 25 442 ± 20 465 ± 21 97 ± 6 96 ± 8
Insulin SC 225 ± 12 237 ± 25 1 25 458 ± 17 482 ± 20 100 100

Cmax: maximum plasma insulin concentration; Tmax: time point of maximum plasma insulin concentration; AUC: area under the plasma insulin concentration versus time
data; RBA: relative bioavailability compared to subcutaneous injection. Superscript number of 1 or 2 indicates at the first or second administration.

Table 3
Pharmacodynamic parameters for plasma glucose levels of diabetic rats after administration of unloaded microneedles (MNs, control), insulin-loaded MNs (0.2 IU), and a
subcutaneous injection of insulin (insulin SC, 0.2 IU) (n = 4).

Group C1min (%) C2min (%) T1min (h) T2min (h) AAC0?6 (% h) AAC24?30 (% h) RPA1 (%) RPA2 (%)
Unloaded MNs 97 ± 8 – 1 – 5±5 – – –
Insulin-loaded MNs 46 ± 10 35 ± 18 1 25 121 ± 9 131 ± 21 96 ± 7 90 ± 11
Insulin SC 42 ± 11 28 ± 8 1 25 126 ± 11 146 ± 18 100 100

Cmin: minimum glucose level; Tmin: time point of minimum glucose level; AAC: area above the plasma glucose level versus time data; RPA: relative pharmacological
availability compared to subcutaneous injection. Superscript number of 1 or 2 indicates at the first or second administration.

insulin was almost completely absorbed from the skin into the sys- array and then integrating them together. The microscopic align-
temic circulation. ment of the MNs and supporting arrays increased the difficulty
of production.
4. Discussion In the current study, we created a dissolving MN patch, com-
prising c-PGA MNs and PVA/PVP supporting structures, by using
MN patches are promising devices for delivering drugs and vac- a simple casting process. The drug-containing c-PGA gel was first
cines into the skin. However, such devices are limited by the total used to fill the mold cavities, and the residual gel on the mold sur-
dose administrated, which is small because of the inherently small face was recycled to avoid wastage. A PVA/PVP solution without
volume and surface area of MNs. Moreover, considerable deforma- drugs was subsequently placed on the first layer and then cen-
tion occurs around the insertion site because of skin elasticity, trifuged to form supporting structures and a backing layer. This
which markedly reduces the insertion depth of the MNs [40,41]. method does not involve a complicated alignment and combina-
Incomplete insertion of the MNs may further result in a reduced tion procedure [42] and is thus suitable for mass production.
dose delivery and wastage of valuable medicines. Similar to the PLA supporting array, the PVA/PVP supporting
In a previous study, we presented an MN delivery system, com- structure was designed to provide an extended length for counter-
posed of chitosan MNs and a poly(L-lactide-co-D,L-lactide) (PLA) acting the skin compressive deformation and mechanical strength
supporting array, for the complete delivery of encapsulated thera- for enabling fully inserting the MNs (P600 lm; Fig. 4). Notably,
peutics [42]. The strong PLA supporting array was designed to pro- when inserted into the skin, both the MNs and supporting struc-
vide mechanical strength for skin insertion. We demonstrated that tures quickly dissolved in the skin within 4 min to deliver the
the chitosan MNs can be fully inserted and embedded within the entire drug load (Fig. 5). The patients do not need to remove the
skin, enabling complete and sustained release of antigens. supporting structures or worry about removing them correctly.
However, after insertion, the PLA supporting array had to be The self-dissolving property of the PVA/PVP supporting structures
removed from the skin for separation from the MNs, causing incon- renders this system user-friendly.
venience to the users. Furthermore, the chitosan-PLA MN array had Previous studies have reported that the onset of hypo-
to be fabricated by separately preparing the MNs and supporting glycemic response was generally slower with the dissolving MN

Please cite this article in press as: M.-C. Chen et al., Poly-c-glutamic acid microneedles with a supporting structure design as a potential tool for transder-
mal delivery of insulin, Acta Biomater. (2015), http://dx.doi.org/10.1016/j.actbio.2015.06.021
10 M.-C. Chen et al. / Acta Biomaterialia xxx (2015) xxx–xxx
administration than with the SC injection [12,16] because of the
time required for MN dissolution. For example, the time required
to reach the minimal plasma glucose level with the starch and
gelatin MN was 3 h, which was significantly longer than that of
the SC group [16]. The slower onset necessitates administering
insulin earlier before meals, which is inconvenient for patients.
In this study, the insulin-loaded c-PGA MN exhibited a rapid onset
of action at 30 min and a maximal/minimal peak action at 1 h,
showing almost no time lag compared with the SC insulin admin-
istration (Fig. 10). This may be attributed to the highly hydrophilic
nature of c-PGA, which results in a quick MN dissolution to release
the encapsulated insulin. Moreover, complete insertion of c-PGA
MNs is beneficial for directly delivering insulin to the dermis layer,
shortening the drug diffusion distance, and resulting in quick
absorption of drugs into the blood capillaries.
After we administered the insulin-loaded c-PGA MNs to dia-
betic rats, the blood glucose levels (Fig. 10b) in the rats was
reduced efficiently and RPA and RBA values in the range of 90–
97% were obtained (Tables 2 and 3), indicating that the MN fabri-
cation process used in this study does not alter the activity of
encapsulated proteins. Furthermore, there were no significant dif-
ferences in the AAC and AUC between the first and second treat-
ments of insulin-loaded MNs (Table 2 and 3). These results
confirmed the efficacy and stability of using c-PGA MNs for insulin
delivery; this system may be an ideal candidate for treating T1DM.
5. Conclusion
This study demonstrated that c-PGA MNs with a supporting
structure design enable complete and efficient delivery of insulin
into the skin. The PVA/PVP supporting structure can counteract
the compressive deformation of the skin and provide mechanical
strength during puncture, thus enabling full insertion of the MNs.
After insertion, both the supporting structures and MNs dissolve
into the skin within 4 min to deliver the entire drug load, without
requiring the users to remove any sharps or waste. These results
suggest that the MN system featuring this unique design enables
a quick convenient self-administration method for delivering pro-
tein drugs and is an attractive alternative to hypodermic needles.
Disclosure
The authors declare no potential conflicts of interest with
respect to authorship.
Acknowledgments
This work was supported by grants from the Ministry of Science
and Technology (MOST 103-2221-E-006-083-MY3 and MOST
103-2622-E-006-034-CC2), and Ministry of Economic Affairs
(TDPA 103-EC-17-A-08-S1-204), Taiwan, Republic of China.
Appendix A. Figures with essential color discrimination
Certain figures in this article, particularly Figs. 1–10 are difficult
to interpret in black and white. The full color images can be found
in the on-line version, at: http://dx.doi.org/10.1016/j.actbio.2015.
06.021.
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