Formulation of Microneedles Coated with Influenza Virus-like Particle Vaccine
Yeu-Chun Kim,1,2Fu-Shi Quan,2Richard W. Compans,2,3Sang-Moo Kang,2,3and Mark R. Prausnitz1,3
Received 15 February 2010; accepted 11 June 2010; published online 30 July 2010
Abstract. Mortality due to seasonal and pandemic influenza could be reduced by increasing the speed of
influenza vaccine production and distribution. We propose that vaccination can be expedited by (1)
immunizing with influenza virus-like particle (VLP) vaccines, which are simpler and faster to
manufacture than conventional egg-based inactivated virus vaccines, and (2) administering vaccines
using microneedle patches, which should simplify vaccine distribution due to their small package size and
possible self-administration. In this study, we coated microneedle patches with influenza VLP vaccine,
which was released into skin by dissolution within minutes. Optimizing the coating formulation required
balancing factors affecting the coating dose and vaccine antigen stability. Vaccine stability, as measured
by an in vitro hemagglutination assay, was increased by formulation with increased concentration of
trehalose or other stabilizing carbohydrate compounds and decreased concentration of carboxymethyl-
cellulose (CMC) or other viscosity-enhancing compounds. Coating dose was increased by formulation
with increased VLP concentration, increased CMC concentration, and decreased trehalose concentration, as
well as increased number of dip coating cycles. Finally, vaccination of mice using microneedles stabilized by
trehalosegenerated strongantibodyresponsesand providedfullprotectionagainsthigh-doselethalchallenge
infection. In summary, this study provides detailed analysis to guide formulation of microneedle patches
coated with influenza VLP vaccine and demonstrates effective vaccination in vivo using this system.
KEY WORDS: coating formulation; influenza; intradermal immunization; microneedle; virus-like
Influenza vaccination is a critical public health measure
to protect against morbidity and mortality related to seasonal
and pandemic influenza (1). Currently, inactivated virus and
live-attenuated virus vaccines are predominantly used and
administered by intramuscular injection using a hypodermic
needle and nasal spray using a special syringe, respectively
(2). The conventional seasonal influenza vaccines are triva-
lent, including two influenza A subtypes (H1N1 and H3N2)
and one variant of influenza B virus (3).
There are, however, limitations to current vaccines and
vaccination methods. Speed of vaccine production and
distribution is very important to effective vaccination,
because antigenic drift requires development of new vaccines
every year to treat seasonal disease and, in some cases, to
address a novel pandemic strain (3). As a result, seasonal
vaccines are manufactured according to a strict 6-month
timetable and then administered to almost 250 million of
patients by medical personnel over the course of a number of
additional months during influenza season (4). The whole
process of seasonal influenza vaccine production and distri-
bution takes close to 1 year. During the recent pandemic of
novel H1N1 “swine” influenza, production was expedited, but
vaccine still did not become available until after 6 months and
vaccine was in short supply for a number of months after that
(5). Thus, there is a need to improve the speed of vaccine
production and distribution. As discussed below, we propose
that development of VLP influenza vaccines can expedite
vaccine production and their administration using a micro-
needle patch can expedite distribution.
Various novel vaccines have been developed such as
recombinant vaccines, DNA vaccines, vaccines developed by
reverse genetics, and subunit vaccines such as virus-like
particles (VLP) (2,6,7). VLPs are attractive because they
can expedite vaccine production and also offer the potential
for improved immunogenicity and safety due to their cell-
based production methods that avoid the delays and safety
concerns of conventional egg-based influenza vaccine pro-
duction (8). Although there are no marketed VLP vaccines
against influenza, there are VLP vaccines against hepatitis B
and human papillomavirus in widespread use (9).
VLP vaccines might especially benefit from administra-
tion using a microneedle patch to expedite distribution to
large populations. Microneedles have been fabricated by
adapting manufacturing tools from the microelectronics
industry to produce micron-scale needles that painlessly
pierce the outer barrier layer of the skin to administer
1School of Chemical and Biomolecular Engineering, Georgia Institute
of Technology, 311 Ferst Dr, Atlanta, Georgia 30332, USA.
2Department of Microbiology and Immunology, Emory University
School of Medicine, Atlanta, Georgia 30322, USA.
3To whom correspondence should be addressed. (e-mail: rcompan@
emory.edu; firstname.lastname@example.org; email@example.com)
AAPS PharmSciTech, Vol. 11, No. 3, September 2010 (#2010)
1530-9932/10/0300-1193/0#2010 American Association of Pharmaceutical Scientists
vaccine in a simple manner using a patch-like format.
Microneedle patches are small and simple enough to use that
they could be distributed through pharmacies or the mail and
might be self-administered by patients (10)
Solid microneedle patches have been coated with
vaccines, including the model antigen ovalbumin (11), hep-
atitis B surface antigen (12), and inactivated influenza virus
(13–15). In addition, hollow microneedles have previously
been used to inject influenza vaccine into the skin of human
subjects (16) and solid microneedles have been used to scrape
the skin for delivery of DNA and other vaccines (17),
although these methods do not lend themselves as easily to
rapid distribution or self-administration.
In addition to simplified logistics, microneedle vaccina-
tion may offer immunologic advantages due to targeting
delivery to the skin, which is replete with antigen-presenting
cells (18). For example, intradermal injection has shown
evidence for dose sparing (19–22) and improved immunoge-
nicity in the elderly (23). However, intradermal vaccination
by conventional methods can be unreliable and requires the
expertise of a trained medical professional (24). In contrast,
microneedles inherently target delivery to the skin using a
simple device, which can ultimately lead to local effects on
depositing vaccines within the skin and systemic effects on
transferring vaccines to the draining lymph nodes through the
vasculature or lymphatics after uptake by antigen presenting
cells such as dendritic and/or Langerhans cells. Animal
studies have shown that microneedles coated with inactivated
influenza vaccine can generate stronger humoral and cellular
immune responses than intramuscular vaccination (25,26).
Microneedles have also been shown to be painless and well
tolerated in humans subjects (27–29).
Building off this body of literature demonstrating
possible advantages of VLP vaccination using microneedles,
this study investigated the effect of vaccine formulation, as
well as the microneedle material and coating process
parameters, on the amount and integrity of influenza VLP
vaccine coated onto microneedles. This is the first detailed
study to examine and optimize coating of a VLP vaccine onto
MATERIALS AND METHODS
Preparation of Influenza VLP Vaccine
VLPs were prepared as described previously (30).
Briefly, to produce VLPs containing influenza virus M1
matrix protein and hemagglutinin (HA), Sf9 cells were co-
infected with rBVs expressing M1 and hemagglutinin
derived from the strain of A/PR/8/34 H1N1 virus. Culture
supernatants were harvested at 3 days post-infection and
purified by low-speed centrifugation (6,000 rpm for 20 min
at 4°C) to remove cells. VLPs in the supernatant were
pelleted by ultracentrifugation (28,000 rpm for 60 min). The
sedimented particles were resuspended in phosphate-buf-
fered saline (PBS) at 4°C overnight and further purified
through a 15-30-60% discontinuous sucrose gradient at
28,000 rpm for 1 h at 4°C. The VLP bands were collected
and analyzed using Western blot and hemagglutination
assays. The size of VLP particles ranges from 80 to
120 nm (30).
For visualization of VLP vaccine delivery by micro-
needles, VLPs were labeled by lipid staining with a red
fluorescent dye. To perform staining, 200 μL VLP at a
concentration of 3 mg/ml was mixed with 10 μL of octadecyl
rhodamine B chloride (R18, Invitrogen, Carlsbad, California,
USA) and incubated at 25°C for 1 h. To remove unbound
R18 molecules, the labeled VLPs were suspended in 10 mL
PBS and precipitated by ultracentrifugation (28,000 rpm for
1 h, Optima L-80 XP, Beckman Coulter, Fullerton, California,
USA) with a 20% sucrose layer (31). The precipitated VLPs
were again washed in PBS by ultracentrifugation to further
remove residual R18 dye.
Fabrication and Coating of Microneedles
Arrays of solid metal microneedles were fabricated by
cutting needle structures from stainless steel sheets (McMaster-
Carr, Atlanta, Georgia, USA) using an infrared laser (Reso-
netics Maestro, Nashua, New Hampshire, USA) as described
previously (32). Individual rows containing five microneedles
were dip coated by horizontally dipping the microneedles into a
coating solution containing 3-8 mg/ml VLP vaccine. The coating
solution was composed of 0.25-1.0% (w/v) carboxymethylcellu-
lose (CMC) sodium salt (USP grade, Carbo-Mer, San Diego,
New Jersey, USA) in PBS as described previously (32,33).
Additional studies used other viscosity-enhancing agents,
including 1% (w/v) alginic acid, polyvinylpyrrolidone, gum
ghatti, karaya gum (Sigma-Aldrich, St. Louis, Missouri,
USA), and xanthan gum (Fluka, Buchs, Switzerland).
Trehalose at a concentration of 5-30% (w/v; Sigma-Aldrich)
was used as a stabilizer in most studies. Additional studies
used other stabilizers, including 15% (w/v) sucrose, glucose,
inulin from dahlia tubers and chicory, and dextrans with
molecular weights of 9 and 36 kDa (Sigma-Aldrich). Unless
otherwise stated, the standard coating solution contained
1% (w/v) CMC sodium salt and 0.5% (w/v) Lutrol F-68 NF
in PBS buffer solution either with or without 15% (w/v)
Imaging and Histology
Microneedles were imaged by scanning electron micro-
scopy (LEO 1530, Carl Zeiss, Oberkochen, Germany), bright
field microscopy (SZX12, Olympus America, Tokyo, Japan)
with a CCD camera (DC 300, Leica Microsystems, Bannock-
burn, Illinois, USA), and fluorescence microscopy (IX70,
Olympus) with a CCD camera (RT Slider, Diagnostic Instru-
ments, Sterling Heights, Michigan). Histological skin sections
were imaged using a Zeiss LSM/NLO 510 Confocal/Multi-
Photon Microscope (Zeiss, Oberkochen, Germany) with a
100× oil-immersion objective used in conjunction with oil
having an index of refraction of 1.51. To image delivery of
viral antigen into skin, microneedles coated with R18-labeled
virus were inserted into human cadaver skin for 10 min and
fixed by freezing in histology mounting compound (Tissue-
Tek®, Sakura Finetek, Torrance, California, USA) for 10 min,
after which microneedles were removed and skin was
sectioned using a cryostat (Cryo-Star HM 560 MV, Microm,
Hésingue, France) for imaging. This use of human skin was
1194 Kim et al.
approved by the Georgia Institute of Technology Institutional
In vitro HA Activity Testing of Influenza VLPs
after Microneedle Coating
We measured HA activity of VLPs to test their stability
after coating onto microneedles. To avoid the more time-
consuming process of coating microneedles, we instead coated
3×3 mm pieces of the same stainless steel used to make
titanium, nickel, copper, glass, polystyrene, and polycarbonate
(Sigma-Aldrich). Coatings were produced by mixing 1 μL of
coating solution with 1 μL of VLP vaccine on the metal piece,
which was allowed to air dry at room temperature overnight.
The metal piece was then dissolved in 100 μL of PBS for 12 h.
Validation experiments showed that VLP HA activity after
coating pieces of stainless steel was similar to that after coating
stainless steel microneedles (data not shown).
To determine HA titers, VLP vaccine dissolved from
metal pieces was serially diluted in 100 μL of PBS deficient in
Mg2+and Ca2+, mixed with an equal volume of a fresh 0.5%
suspension of chicken red blood cells (Lampire Biological
Laboratories, Pipersville, Pennsylvania, USA), and incubated
for 1 h at 25°C. HA titers were determined as the endpoint
dilutions inhibiting the precipitation of red blood cells (34).
In vivo Immunogenicity Testing of Influenza VLPs
After Vaccination Using Microneedles
Balb/c mice (n=4 animals per group, 8-10 weeks old,
female, Charles River Laboratories, Wilmington, Massachu-
setts, USA) were anesthetized intramuscularly with 110 mg/kg
ketamine HCl and 11 mg/kg xylazine. Hair at the site of
treatment was removed by depilatory cream (Nair, Princeton,
washing with a cotton ball soaked with 70% ethanol and drying
with hairdryer, microneedles coated with 4 μg of influenza VLP
vaccine were inserted into the skin on the dorsal surface. In our
previous study, depilatory cream and 70% ethanol was shown
not to effect skin permeability to influenza virus vaccine (35).
Microneedles were coated using the standard coating formula-
for 10 min to allow dissolution of the vaccine antigen from the
At weeks 2 and 4 after vaccination, influenza virus-
specific antibody (IgG) titer was determined using standard
protocols. Briefly, 96-well microtiter plates (Nunc-Immuno
Plate MaxiSorp; Nunc Life Technologies, Basel, Switzerland)
were coated with 100 μl of inactivated A/PR8 virus at a
concentration of 4 μg/ml in coating buffer (0.1 M sodium
carbonate, pH 9.5) at 4°C overnight. After washing, 100 times
diluted serum samples were added into plates and plates were
incubated at 37°C for 1 h. After additional washing, the plates
were then incubated with horseradish peroxidase-labeled
goat anti-mouse IgG (Southern Biotechnology, Birmingham,
Alabama, USA) at 37°C for 1.5 h and then the substrate
ophenylenediamine (Zymed, San Francisco, California, USA)
in citrate-phosphate buffer (pH 5.0) containing 0.03% H2O2
(Sigma-Aldrich) was used to develop color. The optical
density at 450 nm was read using an ELISA reader (model
680; Bio-Rad, Hercules, California, USA).
For challenge infections, isoflurane-anesthetized mice
were infected with 1,000 pfu live influenza viruses (A/PR8/
34, 20×LD50) intranasally in 50 μl of PBS per mouse on
week 5 after vaccination. Naïve mice that received no
vaccination were included as a negative control group. Mice
were observed daily to monitor changes in body weight and
to record death. Mice were euthanized if their body weight
loss exceeded 25% to minimize suffering. All animal experi-
ments and husbandry involved in studies presented in this
manuscript were conducted under the guidelines of the
Emory University Institutional Animal Care and Use Com-
mittee (IACUC). Emory IACUC operates and is conducted
in full compliance with local, national, ethical, and regulatory
principles and local licensing regulations, per the spirit of
Association for Assessment and Accreditation of Laboratory
Animal Care International's expectations for animal care and
Every assay was measured using at least three samples,
from which the arithmetic mean and standard error of the
mean were calculated. A two-tailed Student’s t test (α=0.05)
was performed when comparing two different conditions.
When comparing three or more conditions, a one-way
analysis of variance (ANOVA; α=0.05) was performed. A
value p<0.05 was considered statistically significant.
Fabrication of Microneedles
We fabricated solid stainless steel microneedles by laser
cutting and electropolishing. The microneedles used in this
study were prepared as single rows containing five micro-
needles each measuring 700 μm long, 160 μm wide at the
base, and 50 μm in thickness (Fig. 1a).
Coating and Delivery of Influenza VLP Vaccine
Previous studies have addressed coating of microneedles
with compounds including calcein, vitamin B, bovine serum
albumin, and plasmid DNA (11,32,36), but this is the first
study to examine and optimize coating with a VLP vaccine.
Guided by previous studies, we designed the coating for-
mulation to contain a surfactant (i.e., Lutrol F-68 NF) to
generate uniform coatings by reducing surface tension, a
viscosity enhancer (i.e., CMC) to enable thicker coatings by
increasing coating solution residence time on the microneedle
surface during the drying process, and a stabilizer (i.e.,
trehalose) to prevent the loss of VLP HA activity during
This standard coating formulation was able to coat
influenza VLP vaccine onto microneedles (Fig. 1b). The
coating was thick due to the large amount of stabilizer in the
formulation (i.e., 87% of dissolved solids). Nevertheless,
when coated microneedles were inserted into the skin of
mice, the vaccine coating was efficiently dissolved and
released into the skin almost completely.
1195 Influenza Virus-like Particle Microneedle Vaccination
The speed of VLP vaccine release into skin is also
important. To assess the release kinetics, influenza VLPs
were labeled with a red fluorescent compound and visualized
using fluorescence and multi-photon microscopy. As shown in
Fig. 2a, coated VLP vaccine was efficiently released from
microneedles after insertion into human cadaver skin within
2 min. As a comparison, a VLP-coated microneedle incu-
bated in PBS for 1 h demonstrates complete release.
To assess the localization of VLP vaccine after delivery
into skin, histological sections were prepared after micro-
needle delivery of fluorescently tagged VLP into human
cadaver skin and viewed by multi-photon microscopy. The
representative image in Fig. 2b shows the microneedle
insertion point (white arrow) and the deposition of VLP
vaccine along the needle track in the skin. Some vaccine
appears to have diffused horizontally away from the insertion
site. Altogether, these results indicate that VLPs can be
coated onto the surface of metal microneedles and efficiently
released into the skin.
Finally, we wanted to determine the dose of VLPs that
could be coated onto microneedles. We therefore varied two
process parameters: VLP concentration in the coating sol-
ution and the number of times microneedles were dipped into
the coating solution. As shown in Fig. 3a, increasing the
number of dips increased the dose of VLP coated onto
microneedles in an approximately proportional manner from
0.16 to 0.42 μg per microneedle after increasing from three to
nine dips (ANOVA, p<0.05). Figure 3b shows that increasing
VLP concentration further increased the coated dose in an
approximately proportional manner from 0.42 to 1.1 μg per
microneedle at concentrations of 3 and 7 mg/ml, respectively
(ANOVA, p<0.05). Guided by these data, protocols can be
developed to coat up to more than 1 μg of VLP vaccine per
microneedle, which corresponds to hundreds of micrograms
of vaccine on a patch containing hundreds of coated micro-
needles. Given that VLP vaccines are generally given at doses
well below this (9), coated microneedles appear to be capable
of administering suitable doses of VLP vaccines.
HA Activity of Influenza VLP
VLPs used in this study contain influenza matrix protein
and glycoproteins such as hemagglutinin as described pre-
viously (37). In contrast to chemically inactivated influenza
virus vaccines, VLPs contain unmodified native hemaggluti-
nin antigens. Thus, it is important to protect HA activity
during the drying process when coating microneedles, which
we assessed by measuring red blood cell HA activity of
influenza VLPs after coating and dissolution in PBS.
Drying influenza VLPs in PBS reduced HA activity to
40% relative to the control sample of untreated VLPs
(Fig. 4a). This shows that drying VLPs can damage their
activity. Drying VLPs in our standard coating formulation
containing CMC and a surfactant caused even greater loss of
HA activity to just 2%. Replacing CMC with other viscosity
enhancing excipients, including alginic acid, polyvinylpyrroli-
done, gum ghatti, karaya gum, and xanthan gum, yielded
similar results, although xanthan gum provided somewhat
Fig. 1. Microneedles coated for vaccine delivery. a Scanning
electron micrograph of a microneedle (scale bar 100 μm), b
microneedle array containing five microneedles coated with influ-
enza virus-like particle (VLP) vaccine in standard coating solution
containing trehalose (scale bar 400 μm)
Fig. 2. Influenza VLP vaccine delivery from coated microneedles into skin. a Representative fluorescence micrograph of microneedles coated
with red-fluorescent, R18-stained VLPs (left) and after insertion into human cadaver skin for 30, 60, and 120 s. As a positive control to confirm
complete release of VLPs from the microneedles, microneedles were incubated in PBS for 1 h (right; scale bar 500 μm). b Multiphoton
fluorescence micrograph of cryosectioned human cadaver skin after insertion of R18-stained VLP-coated microneedle (white arrow
microneedle insertion site, scale bar 300 μm)
1196Kim et al.
improved results compared to CMC. However, the coating
dose achieved when using xanthan gum was significantly
lower than that with CMC (data not shown). We therefore
continued to coat using CMC as the viscosity enhancer.
Sugar compounds are known to stabilize influenza viral
vaccines during storage in the dried or frozen state (38) and
previously found to stabilize inactivated influenza virus
during microneedle coating (13). We therefore assessed
whether a set of different stabilizers, including trehalose,
sucrose, glucose, dextran, inulin, could similarly stabilize
influenza VLPs (Fig. 4b). Trehalose was found to retain
100% HA activity of VLPs dried in PBS without CMC (data
not shown) and more than 60% HA activity in the standard
CMC-containing coating solution, which was the best result
among the stabilizers tested. These results indicate that
trehalose is a promising candidate stabilizer for influenza
VLP vaccine coated onto microneedles.
The choice of microneedle material could also affect the
HA activity after coating. We therefore tested seven different
materials including our standard material, stainless steel, as
well as other metals (titanium, nickel, and copper), polymers
(polystyrene, polycarbonate), and glass (Fig. 4c). HA activity
of influenza VLPs coated onto stainless steel was found to be
significantly higher than nickel, copper, titanium, and statisti-
cally indistinguishable from the polymers and glass. To test
the effect of surface roughness on HA activity, stainless steel
Fig. 3. Determination of VLP dose coated onto microneedles. Amount of VLP coated per
microneedle as a function of a the number of dips into coating solution containing 2 mg/mL
VLPs and b the VLP concentration in the coating solution after 6 dips. Standard coating
solution was used. Replicate data from n=4 samples is shown as the mean value with error
bars indicating the standard error of the mean (SEM)
Fig. 4. The effect of microneedle formulation and material on HA activity. HA activity of VLPs is shown after coating and dissolution in PBS.
Coatings were made using a standard coating solution containing 15% trehalose and substituting CMC for various stabilizers (*p<0.05 for
comparison between xanthan gum and other viscous enhancers); b standard coating solution containing various carbohydrate stabilizers at a
concentration of 15% (w/v) (*p<0.05 for comparison between trehalose and other stabilizers); and c microneedles made from various materials
as indicated in the figure legend using standard coating solution containing 15% trehalose (*p<0.05 for comparison between stainless steel and
other metals; n=4, mean±SEM)
1197 Influenza Virus-like Particle Microneedle Vaccination
roughened with sandpaper was compared with untreated
stainless steel. There was no difference in HA activity
between these two conditions.
The Effect of Trehalose Concentration on VLP HA Activity
and Coating Dose
Trehalose was found to play a major role in maintaining
HA activity of influenza VLP vaccine during the microneedle
coating process. To determine the effects of trehalose
concentrations in the coating solution on retaining HA
activity, trehalose concentrations from 0% to 30% were
tested (Fig. 5a). HA activity increased with trehalose concen-
tration (ANOVA, p<0.05). However, addition of trehalose
significantly decreased the dose of VLP vaccine coated onto
microneedles, which makes a direct trade-off between VLP
receptor-binding activity and dose (Fig. 5b). This is probably
because the total amount of material that can be coated onto
a microneedle is limited. Therefore, as more trehalose is
added, there is less room for VLP vaccine. As a compromise,
we have used 15% trehalose throughout the rest of the study
to maintain HA activity while still coating sufficient vaccine
doses onto microneedles.
The Effect of CMC Concentration on VLP HA Activity
and Coating Dose
Because the presence of CMC significantly reduced HA
activity after coating, we studied the effect of CMC concen-
tration in greater detail (Fig. 6). Using a coating solution
containing 0.5% surfactant and 15% trehalose without CMC
retained 100% HA activity (Fig. 6a). The addition of 0.25%
CMC decreased HA activity to 85%. At 0.5-1% CMC,
approximately 50% HA activity was retained. Coating with
1% CMC without trehalose destroyed almost all HA activity.
Altogether, this further shows that CMC significantly dam-
ages HA activity and trehalose can significantly prevent that
To better understand the role of the surfactant, we found
that coating 0.5% surfactant alone retained 41% HA activity
(Fig. 6a). The addition of 15% trehalose to that formulation
restored HA activity to 100%. This indicates that the
stabilizing function of trehalose is not limited to counteracting
the effects of CMC. Therefore, an optimal formulation should
contain surfactant, in order to facilitate good wetting and
spreading of the coating across the microneedle surface, and
should further contain trehalose to retain HA activity.
Although CMC causes functional inactivation of hemag-
glutinin, CMC is an indispensable ingredient for efficient
coating for influenza VLP vaccine. Without CMC, negligible
amounts of VLP vaccine could be coated (Fig. 6b). The dose
of VLP that could be coated increased with CMC concen-
tration from 0% to 1% CMC (ANOVA, p<0.05). HA activity
decreased with increasing CMC concentration from 0% to
0.5% CMC, but showed no further decrease in HA activity
from 0.5% to 1% CMC in the presence of 15% trehalose
(Fig. 6a). These results indicate that 1% CMC is the optimal
condition that balances HA activity loss and coating dose
In vivo Immunogenicity of Influenza VLP Vaccination
Our in vitro studies show that influenza VLP vaccines
can be coated onto microneedles using an optimized for-
mulation to achieve microgram doses and retain most vaccine
HA activity, as measured by HA activity. To validate these
results in vivo, we assessed immunogenicity in mice after
vaccination using microneedles coated with 4 μg of influenza
VLP vaccine formulated using 1% CMC and 0.5% surfactant
either with or without 15% trehalose.
Influenza VLP immunization using microneedles formu-
lated either with or without trehalose significantly increased
virus-specific IgG compared to naïve mice (Fig. 7a). However,
microneedles coated with trehalose induced significantly
higher IgG responses than microneedles without trehalose,
which confirmed the critical role of trehalose to stabilize the
dried VLP vaccine.
Five weeks after vaccination, all mice were challenged
with a lethal dose of A/PR/8/34 influenza virus (20×LD50).
Naïve mice rapidly lost weight and either died or had to be
Fig. 5. The effect of trehalose stabilizer concentration on HA activity and coating dose of
VLPs. a HA activity of VLPs is shown after coating and dissolution in PBS. Coatings were
made using standard coating solution with trehalose added over a range of concentrations
at 25°C. b Trade-off between HA activity and coated dose of VLPs is shown at different
trehalose concentrations (*trehalose concentration, n=4, mean±SEM)
1198Kim et al.
euthanized within 6 days of challenge (Fig. 7b, c). Mice
immunized using microneedles formulated without trehalose
also rapidly lost weight; all of these mice died or were
euthanized within 8 days after challenge. In contrast, mice
immunized using microneedles stabilized with trehalose
showed minimal weight loss, quickly recovered to normal
body weight, and all survived. Altogether, these immunoge-
nicity studies show that microneedle vaccination using
influenza VLPs is effective and that stabilization of the coated
vaccine using trehalose is critically important to retaining
protective efficacy of the vaccine.
Influenza vaccination would benefit from increased
speed of vaccine production and distribution to meet both
seasonal and pandemic needs (3). Influenza VLP vaccines can
increase the speed of vaccine production in part because they
are manufactured using cell culture instead of the cumber-
some egg-based manufacturing methods in use today for
inactivated virus-based vaccines (39). In this study, we
propose that influenza VLP vaccination can be further
expedited by administration using a microneedle patch
applied to the skin. This study carried out the first detailed
examination of influenza VLP vaccine coating onto micro-
needles with the goal of optimizing the formulation to balance
the competing effects of maintaining vaccine immunogenicity
and coating adequate vaccine dose onto the microneedles.
We found that influenza VLP vaccine could be coated
onto microneedles and was then rapidly released into solution
or into the skin. However, VLP vaccine coating onto micro-
needles involves a drying process and requires addition of an
excipient to increase coating solution viscosity for effective
coating. We found that both the drying process and the
inclusion of CMC to increase coating solution viscosity
decreased vaccine activity, as measured by the functional
activity of hemagglutinin. The addition of trehalose to the
coating formulation significantly stabilized the VLP vaccine,
which increased both in vitro HA activity and in vivo
Fig. 6. The effect of CMC concentration on HA activity and coating dose of VLPs. a HA activity and b coating dose of VLPs is shown after
coating and dissolution in PBS. Coatings were made by use of standard coating solution containing 15% trehalose and modified to contain
various concentrtations of CMC. c Trade-off between HA activity and coated dose of VLPs is shown at different CMC concentrations (F-68
Lutrol F-68 NF surfactant, Treh trehalose, *CMC concentration, n=4, mean±SEM)
Fig. 7. Humoral antibody response and protection efficacy after microneedle immunization with influenza VLP vaccine. a Total influenza virus-
specific serum antibody response (IgG) measured 2 and 4 weeks after immunization with VLP vaccine-coated microneedles. (+p<0.01 for
comparison between microneedle and naïve, *p<0.05 for comparison between formulation with trehalose and without trehalose). b Body
weight change and c survival of mice immunized using antigen-coated microneedles after lethal challenge infection (MN microneedle, Treh
1199 Influenza Virus-like Particle Microneedle Vaccination
protective efficacy. Overall, this study demonstrated that
protective microneedle vaccination with influenza VLPs can
be achieved using an optimized formulation containing an
appropriate stabilizer and viscosity enhancer.
Although inclusion of a viscosity-enhancing excipient is
critical for efficient microneedle coating, its presence desta-
bilized influenza VLPs during the drying process. For
example, CMC caused a loss of HA activity of coated
influenza VLP vaccine in a concentration dependent manner
up to 0.5% concentration in the coating solution. The VLP
HA activity of undried coating solution in the liquid state was
not affected by inclusion of CMC or other viscosity enhancers
in the formulation (data not shown), which suggests that
CMC does not interfere with the HA assay itself. A possible
explanation for CMC’s adverse effects is that high molecular
weight viscosity enhancers might facilitate aggregation of the
VLP particles or physically block the hemagglutinin mole-
cules on the surface of influenza VLPs during drying. We
found that coating microneedles with inactivated influenza
virus also led to loss of HA activity, which was associated with
increased virus particle size probably due to aggregation (33).
In addition to the choice of coating excipients, the choice of
microneedle material also influenced loss of HA activity. While
a number of materials performed similarly to stainless steel, we
found that nickel and especially copper caused additional
damage to coated VLPs, although we do not know mechanism
by which these materials help deactivate HA activity.
The addition of sugar molecules significantly improved
influenza VLP stability by preserving HA activity during the
drying process.Inparticular, theaddition of trehalosepreserved
approximately 50-60% in the presence of CMC after drying.
Carbohydrates have previously been shown to protect the
structural integrity of soluble hemagglutinin during lyophiliza-
tion, perhaps due to the formation of an amorphous glassy state
after freeze drying, as shown by differential scanning calorim-
etry analysis (40). Other studies have similarly shown that the
activity of various influenza vaccine was similarly retained after
freeze drying in the presence of carbohydrates, including
trehalose, dextran, and inulin (38,41,42). In contrast, our study
showed that trehalose protected HA activity of influenza VLPs
better than dextran and glucose and much better than inulin
during the air drying process used here. Amorij et al. reported
that visible aggregates were detected ina hemagglutininvaccine
lyophilized in PBS without carbohydrate after reconstitution
(40). Thus, we hypothesize that influenza VLPs may similarly
have been protected by trehalose during drying by preventing
aggregation of VLP particles.
We elected to assess in vitro HA activity of influenza VLPs
through the HA assay. In contrast, other studies have assessed
conformational changes of soluble hemagglutinin subunit vac-
cines by fluorescence and circular dichroism spectroscopy and
found changes in secondary helical conformation and tertiary
structure of hemagglutinin upon freezing in PBS (40). However,
unlike soluble hemagglutinin protein, VLPs resemble the wild-
type virus in structure and morphology. For example, influenza
VLPs contain hemagglutinin glycoprotein trimers embedded
within a lipid bilayer membrane (30). Hemagglutinin is an
important protective target molecule in influenza vaccine (43).
Thus, the stability of influenza vaccine is dependent on
maintaining the native conformation and structural integrity of
hemagglutinin. Any changes in hemagglutinin structure or
conformation could result in loss of receptor binding function
of hemagglutinin as represented by hemagglutination of red
blood cells. Because influenza VLPs are a multi-component
supramolecular particles maintaining the native structure of
hemagglutinin in a membrane-anchored form, determination of
functional activities by HA assay is more appropriate for
monitoring influenza vaccine stability than, for example,
As evidence for this, HA activity of influenza VLPs coated
onto microneedles without trehalose was extremely low. Corre-
spondingly, virus-specific antibodies were only weakly elevated
and immunization without trehalose provided poor protection
against lethal infection. In contrast, influenza VLP vaccine
stabilized using trehalose had much higher HA activity and
correspondingly generated much higher antibody levels and
provided fully protective immunity, as measured by 100%
survival and minimal weight loss after challenge. Therefore,
HA activity in vitro appears to be an indicator of influenza
vaccine integrity that can predict vaccine efficacy in vivo.
This study was motivated in part by the expected logistical
advantages that microneedles can offer to expedite influenza
vaccination. Storage, stockpiling, distribution, and disposal
should be simplified by microneedles, because microneedle
patches are small (i.e., much smaller than a hypodermic needle
and syringe). The possibility of self-administration and distribu-
tion by mail could enable vaccination of large populations (e.g.,
a whole country) within a matter of days. Self-vaccination may
be possible because microneedles are painless (28), do not
require reconstitution, and are simple to apply to the skin.
Altogether, these features of microneedles could increase
coverage of influenza vaccination.
This study provides the first detailed examination of
coating microneedles with influenza VLP vaccine. We found
that VLPs can be coated in microgram quantities suitable for
vaccination. However, drying during the coating process,
especially in the presence of viscosity enhancers such as CMC,
significantly damaged the VLP antigen, as measured by loss of
HA activity in vitro and weaker antibody responses and poor
protection in vivo. Improvement of the coating formulation,
especially through theaddition of trehalose, protected VLP HA
activity in vitro and protective immunity in vivo. Overall, this
study shows that microneedles can be coated with influenza
VLP vaccine to provide adequate dose and immunogenicity for
effective vaccination, as determined in mice.
This work was supported in part by NIH grants R01-
EB006369 (M.R.P.) and U01-AI0680003 (R.W.C.), SERCEB
(R.W.C) and the Georgia Research Alliance Program grant
(S.M.K). We thank Dr. Vladimir Zarnitsyn for microneedle
fabrication, Dr Young-Bin Choy for SEM imaging, and Dr.
Mark Allen for use of laser microfabrication facilities. M.R.P.
serves as a consultant and is an inventor on patents licensed
to companies developing microneedle-based products. These
possible conflicts of interest have been disclosed and are
being managed by Georgia Tech and Emory University.
Kim et al.
REFERENCES Download full-text
1. Ellebedy AH, Webby RJ. Influenza vaccines. Vaccine. 2009;27:
2. Hoelscher M, Gangappa S, Zhong WM, Jayashankar L,
Sambhara S. Vaccines against epidemic and pandemic influ-
enza. Expert Opin Drug Deliv. 2008;5:1139–57.
3. Nichol KL, Treanor JJ. Vaccines for seasonal and pandemic
influenza. J Infect Dis. 2006;194:S111–8.
4. Gerdil C. The annual production cycle for influenza vaccine.
5. Centers for Disease Control and Prevention. Use of influenza
A (H1N1) 2009 monovalent vaccine, recommendations of the
Advisory Committee on Immunization Practices. Morb Mort
Wkly Rep. 2009;58:1–8.
6. Nicholson KG, Wood JM, Zambon M. Influenza. Lancet.
7. Palache AM. Influenza vaccines—a reappraisal of their use.
8. Jennings GT, Bachmann MF. Coming of age of virus-like particle
vaccines. Biol Chem. 2008;389:521–36.
9. Noad R, Roy P. Virus-like particles as immunogens. Trends
10. Prausnitz MR, Mikszta JA, Cormier M, Andrianov AK.
Microneedle-based vaccines. Curr Top Microbiol Immunol.
11. Matriano JA, Cormier M, Johnson J, Young WA, Buttery M,
Nyam K, et al. Macroflux (R) microprojection array patch
technology: a new and efficient approach for intracutaneous
immunization. Pharm Res. 2002;19:63–70.
12. Andrianov AK, DeCollibus DP, Gillis HA, Kha HH, Marin A,
Prausnitz MR, et al. Poly[di(carboxylatophenoxy)phosphazene]
is a potent adjuvant for intradermal immunization. Proc Natl
Acad Sci USA. 2009;106:18936–41.
13. Kim YC, Quan FS, Yoo DG, Compans RW, Kang SM, Prausnitz
MR. Enhanced memory responses to seasonal H1N1 influenza
vaccination of the skin with the use of vaccine-coated micro-
needles. J Infect Dis. 2010;201:190–8.
14. Koutsonanos DG, Martin MdP, Zarnitsyn VG, Sullivan SP,
Compans RW, Prausnitz MR, et al. Transdermal influenza
immunization with vaccine-coated microneedle arrays. PLoS
15. Zhu QY, Zarnitsyn VG, Ye L, Wen ZY, Gao YL, Pan L, et al.
Immunization by vaccine-coated microneedle arrays protects
against lethal influenza virus challenge. Proc Natl Acad Sci
16. Van Damme P, Oosterhuis-Kafeja F, Van der Wielen M,
Almagor Y, Sharon O, Levin Y. Safety and efficacy of a novel
microneedle device for dose sparing intradermal influenza
vaccination in healthy adults. Vaccine. 2009;27:454–9.
17. Mikszta JA, Alarcon JB, Brittingham JM, Sutter DE, Pettis RJ,
Harvey NG. Improved genetic immunization via micromechan-
ical disruption of skin-barrier function and targeted epidermal
delivery. Nat Med. 2002;8:415–9.
18. Debenedictis C, Joubeh S, Zhang GY, Barria M, Ghohestani RF.
Immune functions of the skin. Clin Dermatol. 2001;19:573–85.
19. Auewarakul P, Kositanont U, Sornsathapornkul P, Tothong P,
Kanyok R, Thongcharoen P. Antibody responses after dose-
sparing intradermal influenza vaccination. Vaccine. 2007;25:659–63.
20. Belshe RB, Newman FK, Cannon J, Duane C, Treanor J, Van
Hoecke C, et al. Serum antibody responses after intradermal
vaccination against influenza. New Engl J Med. 2004;351:2286–94.
21. Kenney RT, Frech SA, Muenz LR, Villar CP, Glenn GM. Dose
sparing with intradermal injection of influenza vaccine. New
Engl J Med. 2004;351:2295–301.
22. Hinman AR, Orenstein WA, Santoli JM, Rodewald LE, Cochi
SL. Vaccine shortages: history, impact, and prospects for the
future. Annu Rev Public Health. 2006;27:235–59.
23. Holland D, Booy R, De Looze F, Eizenberg P, McDonald J,
Karrasch J, et al. Intradermal influenza vaccine administered
using a new microinjection system produces superior immuno-
genicity in elderly adults: a randomized controlled trial. J Infect
24. Lambert PH, Laurent PE. Intradermal vaccine delivery: will
new delivery systems transform vaccine administration? Vac-
25. Kim YC, Quan FS, Yoo DG, Compans RW, Kang SM,
Prausnitz MR. Improved influenza vaccination in the skin
using vaccine coated-microneedles. Vaccine. 2009;27:6932–8.
26. Quan FS, Kim YC, Yoo DG, Compans RW, Prausnitz MR,
Kang SM. Stabilization of influenza vaccine enhances protec-
tion by microneedle delivery in the mouse skin. PLoS ONE.
27. Bal SM, Caussin J, Pavel S, Bouwstra JA. In vivo assessment of
safety of microneedle arrays in human skin. Eur J Pharm Sci.
28. Gill HS, Denson DD, Burris BA, Prausnitz MR. Effect of
microneedle design on pain in human volunteers. Clin J Pain.
29. Haq MI, Smith E, John DN, Kalavala M, Edwards C, Anstey
A, et al. Clinical administration of microneedles: skin puncture,
pain and sensation. Biomed Microdevices. 2008;11:35–47.
30. Quan FS, Huang CZ, Compans RW, Kang SM. Virus-like particle
vaccine induces protective immunity against homologous and
heterologous strains of influenza virus. J Virol. 2007;81:3514–24.
31. Ludwig K, Korte T, Herrmann A. Analysis of delay times of
hemagglutinin-mediated fusion between influenza-virus and
cell-membranes. Eur Biophys J. 1995;24:55–64.
32. Gill HS, Prausnitz MR. Coated microneedles for transdermal
delivery. J Control Release. 2007;117:227–37.
33. Kim YC, Quan FS, Compans RW, Kang SM, Prausnitz MR.
Formulation and coating of microneedles with inactivated
influenza virus to improve vaccine stability and immunogenicity.
J Control Release. 2010;142:187–95.
34. Vyas GN, Shulman NR. Hemagglutination assay for antigen
and antibody associated with viral hepatitis. Science.
35. Skountzou I, Quan FS, Jacob J, Compans RW, Kang SM.
Transcutaneous immunization with inactivated influenza virus
induces protective immune responses. Vaccine. 2006;24:6110–9.
36. Chen XF, Prow TW, Crichton ML, Jenkins DWK, Roberts MS,
Frazer IH, et al. Dry-coated microprojection array patches for
targeted delivery of immunotherapeutics to the skin. J Control
37. Quan FS, Steinhauer D, Huang C, Ross TM, Compans RW, Kang
S-M. A bivalent influenza VLP vaccine confers complete inhibition
of virus replication in lungs. Vaccine. 2008;26:3352–61.
38. Amorij JP, Huckriede A, Wischut J, Frifflink HW, Hinrichs WLJ.
Development of stable influenza vaccine powder formulations:
challenges and possibilities. Pharm Res. 2008;25:1256–73.
39. Quan FS, Compans RW, Nguyen HH, Kang SM. Induction of
heterosubtypic immunity to influenza virus by intranasal
immunization. J Virol. 2008;82:1350–9.
40. Amorij JP, Meulenaar J, Hinrichs WLJ, Stegmann T, Huckriede
A, Coenen F, et al. Rational design of an influenza subunit
vaccine powder with sugar glass technology: preventing con-
formational changes of haemagglutinin during freezing and
freeze drying. Vaccine. 2007;25:6447–57.
41. de Jonge J, Amorij JP, Hinrichs WLJ, Wiischuta J, Huckriedea
A, Frijlinkb HW. Inulin sugar glasses preserve the structural
integrity and biological activity of influenza virosomes during
freeze-drying and storage. Eur J Pharm Sci. 2007;32:33–44.
42. Maa YF, Ameri M, Shu C, Payne LG, Chen DX. Influenza
vaccine powder formulation development: spray freeze drying
and stability evaluation. J Pharm Sci. 2004;93:1912–23.
43. Steinhauer DA. Role of hemagglutinin cleavage for the
pathogenicity of influenza virus. Virology. 1999;258:1–20.
1201Influenza Virus-like Particle Microneedle Vaccination