Dissolving Microneedle Patches for Dermal Vaccination
Dissolving Microneedle Patches for Dermal Vaccination
M. Leone 0 1
J. Mönkäre 0 1
J. A. Bouwstra 0 1
G. Kersten 0 1
CD CpG ODN DAMPs DAB dDCs DEPA 0 1
0 Department of Analytical Development and Formulation, Intravacc Bilthoven , the Netherlands
1 Division of Drug Delivery Technology , Cluster BioTherapeutics , Leiden Academic Centre for Drug Research, Leiden University Einsteinweg 55 , P.O. Box 9502, 2300 RA Leiden , the Netherlands
2 J. A. Bouwstra
The dermal route is an attractive route for vaccine delivery due to the easy skin accessibility and a dense network of immune cells in the skin. The development of microneedles is crucial to take advantage of the skin immunization and simultaneously to overcome problems related to vaccination by conventional needles (e.g. pain, needle-stick injuries or needle re-use). This review focuses on dissolving microneedles that after penetration into the skin dissolve releasing the encapsulated antigen. The microneedle patch fabrication techniques and their challenges are discussed as well as the microneedle characterization methods and antigen stability aspects. The immunogenicity of antigens formulated in dissolving microneedles are addressed. Finally, the early clinical development is discussed.
antigen stability; dissolving microneedle fabrication; dissolving microneedle characterization; skin immunization; vaccine delivery
AF4 Asymmetrical flow field–flow
APCs Antigen presenting cells
J. A. Bouwstra and G. Kersten contributed equally to this work.
Vaccination is one of the most successful medical
interventions in history, reducing mortality and morbidity for
several infectious diseases to almost zero in areas where
vaccines are being used (
). Most vaccines are
administered intramuscularly or subcutaneously (Fig. 1) by
injection that may cause pain and discomfort and avoidance
by people with needle-phobia (
). Furthermore, the
hypodermic needles used to administer the vaccine by these
routes generates hazardous waste and can lead to needle
stick-injuries and needle re-use. The latter can spread
infectious diseases such as Hepatitis B and AIDS
particularly in the developing countries (
). Furthermore, the use of
innovative vaccine delivery systems could offer several
other advantages such as antigen thermostability, fewer
booster immunizations and, as a consequence, increase
of the vaccination adherence and a reduced burden on
healthcare personnel. These latter advantages would
especially be beneficial in mass vaccination campaigns, such
as in case of outbreaks, when feasible and fast
immunizations schemes are necessary (
Since the skin is a very immune-competent organ and
easily accessible, dermal vaccine delivery is an attractive
alternative. The viable epidermis and dermis contain
m a n y a n t i g e n p r e s e n t i n g c e l l s ( A P C s ) s u c h a s
Langerhans cells (LCs) and dermal dendritic cells (dDCs)
(Fig. 1) (
). These APCs capture antigens and
subsequently migrate to the draining lymph nodes to present
the antigen to the T-cells to activate Ag-specific T-cells
and B-cells for systemic immune response. Besides LCs
and dDCs, epidermal keratinocytes are also involved in
the immune response by producing cytokines and
chemokines (e.g. TNF-α and IL-1β) to enhance
maturation of APCs and migration to the lymph nodes (11).
Although the skin surface is easily accessible, the skin (Fig.
1) is designed to protect the human body against entry of
foreign organisms or toxic substances (
). Therefore, the
top-layer of the skin, the stratum corneum (in humans 15–
20 μm thick), forms a significant physical barrier for vaccine
delivery. Consequently, the delivery of high-molecular weight
(>500 Da) compounds such as antigens require methods
enabling their penetration into the skin (13). Several methods
s u c h a s p o w d e r a n d f l u i d j e t i n j e c t i o n , t h e r m a l
microporation, sonoporation, transfollicular delivery and
) have been proposed to deliver antigens into
the skin. Recently, microneedles (MNs) have gained great
attention for dermal vaccination. MNs are needle-like
microstructures, up to 1 mm in length (
), typically assembled in
variable numbers on a patch. They pierce the stratum
corneum and underlying tissue to deliver the antigen into
the epidermis or dermis while they are short enough not to
reach pain receptors and thus pain sensation can be avoided
). Furthermore, the immunization with MNs may not
require the healthcare personnel (
) and does not generate
sharp needle wastage after immunization.
The first microneedles were conceptualized for drug
delivery in 1976 (
) but only during the last 20 years
microneedles have been actively developed. MNs can be
classified in the following groups: hollow, coated, porous,
hydrogel-forming, dissolving microneedles (dMNs) and
MNs for pretreatment (
). dMNs consist of
fastdissolving materials (e.g. polymers or sugars) as a matrix
material and the drug/antigen is mixed in the matrix.
After insertion into the skin, they dissolve releasing
simult a n e o u s l y t h e a c t i v e p h a r m a c e u t i c a l i n g r e d i e n t
The scope of this review is to evaluate the use of dMNs
as vaccine delivery systems to overcome the limitations of
traditional subcutaneous (s.c.), intramuscular (i.m.) or
intradermal (i.d.) injections. Preparation methods for dMNs,
their characterization and immunological properties will
be described underlining the potential and novelty of this
MATERIALS AND MANUFACTURING
Matrix material should possess the following
characteristics: biocompatible, biodegradable, low toxicity, strength/
toughness and cheap (
). Many materials have been
used to produce dMNs ( Table I) . H e a d t o h e a d
Fig. 1 Schematic representation of
microneedle insertion and
subcutaneous and intradermal)
injections onto the human skin are
shown. Microneedles penetrate the
stratum corneum reaching the
viable epidermis. The hypodermic
needles puncture the skin during
insertion into the subcutaneous or
muscle tissues. Adapted from (3).
comparisons of the materials used for dMN production
have not been reported as far as we know. The selection
of the matrix material may be based on practical
considerations rather than rational design. Apart from safety,
factors to consider include obtaining MNs capable to
pierce the skin, compatibility with the active compound,
compatibility with the manufacturing procedure
(acceptable viscosity before drying or spraying and reasonable
solidification time) and a potential to scale-up of dMN
patches for mass production (
). The most frequently
used matrix materials are sodium hyaluronate, that is
naturally present in the skin, and sodium
). Both are approved as inactive
materials by FDA for parenteral drug products. Other
materials include poly(vinylalcohol) (PVA) (42),
poly(vinylpyrrolidone) (PVP) (
anhydride (PMVE/MA) (Gantrez AN-139®) (
) and low
molecular weight sugars like maltose (
) and trehalose
). dMNs have also been prepared from biodegradable
polymers such as polylactic-co-glycolic acid (PLGA) (
polylactic acid (PLA) (
) and polyglycolic acid (PGA) (
However, due to their slow dissolution rate in skin and a
preparation method using high temperatures (
organic solvents, these polymers are less suitable as matrix
material. The back-plate of the dMN patch can be made by using
the same (
) or different materials (
) as the needles.
Furthermore, the back-plate can be reinforced or the ease of
handling can be increased by applying an adhesive tape
). Besides matrix material, other excipients
might be included ((33) (
)) to improve the antigen
stability or mechanical strength of the dMNs (Table I).
Antigens that have been used include almost all
vaccine types, ranging from peptides and proteins (
DNA vectors encoding antigenic proteins (
attenuated or inactivated viruses (
). Antigens are
g e n e r a l l y d i sp e r se d di r e c t l y i n th e d M N m a t r ix
) but they can also be encapsulated in
nanoparticles or in a cross-linked structure (
potentiate or alter the immune response (
Furthermore, adjuvant can be incorporated in the
The most common fabrication method of dMNs is
micromolding in which dMNs are prepared using a
polydimethylsiloxane (PDMS) mold (Fig. 2). First, the PDMS
mold is typically produced from a silicon or metallic
master mold (
) that is obtained by using techniques such as
), lithography (
), thermal drawing (
laser micromachining (
). PDMS is a hydrophobic
flexible material, which can very accurately reproduce
the master structure as a negative template (17). The
mold can be re-used for dMN fabrications after
appropriate cleaning. The first step in preparing dMNs using the
PDMS mold is the addition of the polymer/antigen
CpG ODN CpG oligodeoxynucleotides, DT diphtheria toxoid, EV71 Enterovirus 71, Gantrez® AN-139 copolymer of methylvinylether-co-maleic anhydride
(PMVE/MA), HIV human immunodeficiency virus, HBV hepatitis B virus, IPV inactivated polio vaccine, MPLA monophosphoryl lipid A, NPs nanoparticles, Na-CMC
Sodium carboxymethylcellulose, OVA ovalbumin, PAA poly(acrylic acid), PLGA poly-D,L-lactide-co-glycolide, poly(I:C) polyinosinic-polycytidylic acid, PVA
poly(vinylalcohol), PVP poly(vinylpyrrolidone), TT tetanus toxoid
mixture in the mold. This is typically done manually at
the research setting but the mold can also be filled by
using an atomized spray (
). After filling of the mold,
vacuum and/or centrifugation steps are performed to fill
the PDMS microcavities with the polymer/antigen
). Finally, the solution in the mold is dried at
slightly elevated temperature (
). The drying step
c a n b e r e p l a c e d b y p h o t o p o l y m e r i z a t i o n i f
photocrosslinkable material is used (60).
The micromolding can be a straightforward
technique in the laboratory because it requires little
additional equipment. Furthermore, the absence of harsh
conditions (e.g. high temperature or organic solvents) is
an advantage when working with sensitive antigens (
However, it might not be suitable for industrial scale-up
or continuous manufacturing if steps such as manual
removal of air bubbles from the microcavities after
vacuum or centrifugation are needed or if the production
method will result in too much vaccine wastage (see
Antigen Wastage section).
Drawing Lithography. This technique is based on extensional
(stretching) deformation of polymeric material from a
2dimensional to a 3-dimensional structure. Melted polymer is
dispensed on a fixed plate and elongated by drawing pillars in
the upper-moving plate (Fig. 2) (
). The polymer viscosity
is progressively increased by cooling until the glass transition
temperature of the polymer is reached. Finally, further cooling
induces a solid polymer providing the suitable dMN strength
for the skin piercing (
). The advantage of this fast
fabrication method is the minimal polymer wastage due to the
dispensed drops on the plate. However, only a limited number
of polymers have suitable glass transition temperatures for this
). More importantly, this technique is not
appropriate for thermolabile antigens because melting and
transition temperatures are high during the manufacturing (e.g. for
maltose >95°C (
Soft Lithography. In soft lithography dMNs are fabricated
by first pairing a polymer film with the mold with
microcavities and passing them through a heated nip.
Next, the filled mold is placed on a flexible,
watersoluble substrate and passed through the heated nip.
After separation of the mold, a dMN patch on the
substrate remains (Fig. 2). Instead of heated nip, photocuring
can be also used (
). Similarly to drawing lithography,
this manufacturing method claims excellent scalability,
low cost and short preparation time. However, the high
temperature used for the fabrication can be still critical
while using a thermolabile antigen mixed with the
Droplet-Born Air Blowing and dMN on an Electrospun Pillar Array
In droplet-born air blowing (DAB), a droplet of polymer
solution without drug and another droplet of drug
solution are dispensed together on two plates. The upper
plate is moved downwards so that the droplets are
touching and thereafter plates are withdrawn to a distance
corresponding to the two dMN lengths of the lower and
upper plate (Fig. 2). The polymer solutions are dried with
air flow producing a dMN patch on each plate (Fig. 2)
). The advantages include low temperature (4–25°C)
and fast (≤ 10 min) fabrication and minimal drug and
A variant of DAB is dMN on an electrospun pillar array
(DEPA). The flat plate is replaced here by a pillar array
covered by a fibrous sheet. Then, polymer formulation droplets
are dispensed on the pillar array and placed in contact with a
PDMS slab to pull and elongate the droplets obtaining
microneedles (Fig. 2). Finally, elongated droplets are dried
by air flow.
General Challenges of dMN Preparation
Dermal vaccination is attractive especially for the antigen
dose sparing to evoke an immune response. However, the
optimization of the manufacturing methods is crucial to
reduce antigen wastage. During micromolding part of the
antigen is lost in the PDMS mold due to low volume filling
of the microcavities relative to the system volume needed
). It is often mentioned that excess of solution from the
mold can be collected in order to recycle (
However, the saved antigen amount is often not reported
in the literature and more importantly the quality of the
recovered antigen may be difficult to guarantee hampering
reuse of the vaccine formulation.
One possibility to reduce the antigen loss during the
micromolding is to use polymer/antigen solution only
for the dMNs and to produce a backplate only from the
matrix material or even from other material. The
backplate material should possess higher viscosity than that of
the needles to reduce the diffusion of the antigen from the
dMNs during preparation and drying (
). In stability
studies presence of antigen in the needles and its absence
in the backplate should be monitored to demonstrate lack
of diffusion of antigen to the backplate during storage
). However, in literature this aspect is generally not
addressed. In fabrication methods like drawing
lithography, DAB and DEPA, the antigen is dispensed in drops,
thus the antigen wastage can be potentially reduced
drastically. However, it is not reported if antigen can be lost in
the dispensing instrument.
dMN tips. This can be particularly challenging in the case of
antigens encapsulated in nanoparticles, an approach to
improve immunogenicity of dermally delivered subunit antigens
). Another aspect to consider is the delivery efficiency, i.e.
the relation between the antigen amount incorporated into
the dMNs and the antigen dose actually delivered into the
skin. Unfortunately, these aspects are often not described in
detail in the literature, although systems have been and are in
development to maximize delivery (see next section). This
makes comparison of different concepts difficult if not
impossible. An additional issue is the physico-chemical properties of
the adjuvant, that determines whether the adjuvant can be
mixed properly with the matrix material.
Fabrication Aimed to Improve Delivery Efficiency
In order to facilitate the delivery of the entire intended
antigen dose into the skin, some modified fabrications
have been developed. These include micromolding of
arrowhead dMNs mounted on mechanically strong shafts
) or dMNs presenting an elongated base increasing
the needle length (51). Drawing lithography has been
modified by dispensing melted polymer on a fixed plate
presenting pedestals (
). DEPA presents patch pillars to
improve the delivery efficiency. After patch application
into the skin, dMNs separate from the pillars due to a
tensile breaking force of the fibrous sheet between the
pillar and the dMN (Fig. 2). This allows a proper
implantation of the dMNs into the skin and removal of the
remaining back plate without the need to wait dMN
Other critical steps during the dMN preparation are related to
the high temperature reached in some manufacturing
methods. The micromolding usually is done at mild
temperatures. However, when using methods such as drawing and
soft lithography, temperatures around 100°C may be
required. Such a temperature can be critical when using
thermolabile antigens mixed to the matrix. Alternatively,
photocurable polymers like acrylate-based polymers (
poly (ethylene glycol) diacrylate (PEGDA) (
) may be
used. However, radiation should not damage proteins or
DNA of vaccines. In all methods, a drying step is included
which can be detrimental for protein antigens even at
moderate temperatures (
Antigen and Adjuvant Loading
Besides reproducible loading (
) and dose homogeneity,
dMNs should contain a sufficient high antigen and adjuvant
dose, which can be challenging due to very low volumes of
Because dMNs deliver antigen into the viable skin, they
should be sterile and have low endotoxin content (
Since the final product is dry, a sterile filtration step, if
at all possible, should be done on final fluid bulk,
implying that the actual patch manufacturing should be
performed under aseptic conditions (
sterilization of patches by gamma irradiation may be
considered, although this can damage the antigen (
and may be difficult to validate. Based on FDA
guidelines for medical devices in direct contact with
lymphatic tissue, the endotoxin content in dMNs should be
CHARACTERIZATION OF DISSOLVING
A number of unique parameters must be determined to
assess dMN quality (Table II). Since there are no licensed
products on the market, no MN monographs exist in
). Below, aspects that may be of
importance are discussed.
Shape and sharpness of MNs are typically investigated by
microscopic techniques such as light and scanning electron
). During product development,
microscopy can be used also to analyze the distribution of
fluorescent-labelled antigen in the MNs (
dMNs are dry formulations and it is important to
measure their water content by using methods such as Karl
Fisher titration (a coulorimetric or volumetric titration to
determine trace amounts of water in the sample),
thermogravimetric analysis or moisture balance (
water content can influence mechanical properties,
protein stability and dissolution kinetics (
). The generally
recommended water content for freeze-dried vaccines is
less than 3% (w/w) (
), that could be also taken as
guideline for dMNs.
Stability of the antigen should be assessed both after the
manufacturing of dMNs (
) as well as after the storage
). The type of stability indicating assays
depends on the antigen as well as the type of immunity that
should be induced (e.g. for antibody responses the tertiary
structure of protein is important). Protein conformation
can be assessed by spectroscopic techniques such as
Antigen distribution in
Skin piercing efficiency
Dissolution of MNs
Antigen localization into
Stability after storage
AF4 asymmetrical flow field–flow fractionation, CD circular dichroism, DLS dynamic light scattering, ELISA enzyme-linked
immunosorbent assay, HP-SEC size exclusion chromatography, MFI micro-flow imaging, NTA nanoparticle tracking
analysis, SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SRID single radial immunodiffusion assay,
TEM transmission electron microscopy, UV-Vis ultraviolet–visible spectroscopy
circular dichroism (
) and fluorescence spectroscopy (
Protein backbone integrity can be analyzed also by
). However, this method is not suitable to
examine the protein unfolding, indicating the loss of B-cell
epitopes. The obvious way to analyse B-cell epitopes is by
measuring antigenicity with immunoassays such as
ELISA. In case of incorporation of DNA in dMNs, agarose
gel electrophoresis and in vitro transfection can be
performed to measure the DNA supercoiling and efficacy
The aggregation of protein antigens or particulate
vaccines can be investigated by several methods such as size
exclusion chromatography (HP-SEC) (
flow field–flow fractionation (AF4) (
imaging (MFI) (
), transmission electron microscopy (TEM)
), dynamic light scattering (DLS) (
) and nanoparticle
tracking analysis (NTA). For live attenuated or vector
vaccines the viability of virus or bacterium may be sufficient
because the antigen will replicate after immunization and
so the vaccine potency can be determined by measuring
the titer of live antigen (
). Finally, immunogenicity
studies are crucial to determine vaccine potency (
limiting factor for characterization and quality control may
be the small sample sizes and matrix effects due to high
concentrations of matrix component after dissolution of
So far, a few studies have systematically analyzed
vaccine stability in dMNs. Mistilis et al. showed that the
buffer composition and preparation conditions (e.g. drying
temperature) must be carefully selected to retain the
vaccine stability of subunit influenza vaccine (
analysis of hemagglutinin activity showed that ammonium
acetate buffer (pH 7.0) and HEPES retained the
antigenicity much better in solution and dry state than when
using phosphate-buf fers. In add ition, surf actants
destabilized the antigen especially in liquid formulation
prior to dMN fabrication and they may cause
crystallization of the MN matrix damaging the antigen (
encapsulation plays also a role in the antigen stabilization.
Similar antigen-specific CD8+ proliferative responses for
OVA-PLGA NPs in dMNs before and after 10 weeks
storage at ambient conditions were obtained (
contrast, groups immunized with 10 weeks stored monomeric
OVA in dMNs showed a decrease in T-cell response in
comparison with the group immunized with non-stored
dMN Mechanical Strength and Skin Penetration
The mechanical properties of MNs (e.g. strength or facture
force) should be analyzed to determine whether dMNs are
strong enough and do not fracture during skin penetration
), unless it is intended so. Measurements of dMN
d i s p l a c e m e n t - f o r c e c a n b e p e r f o r m e d b y u s i n g a
displacement-force test station to compare different matrix
materials or geometry (
) or the effect of storage
conditions (24). Subsequent skin penetration studies are typically
analyzed on ex vivo human (
) or porcine skin (
However, it is also important to consider the in vitro-in vivo
correlation of the subcutaneous layers as these layers can also
affect microneedle performance. For this purpose, artificial
gel-layers can be used to resemble the in vivo situation more
). After MN application and removal from the skin,
the skin is stained with dye (e.g. trypan blue). Additionally,
stratum corneum can be stripped and the number of
penetrating tips per patch can be determined. The penetration of
single MN through the skin layers can be examined in a
detailed way by analysing histological cross-sections of skin,
although this is a more laborious approach (
) and not
suitable for routine analysis. The depth of deposition of
fluorescently-labelled antigen in the skin can be investigated
by confocal microscopy (
) or fluorescence microscopy by
using skin cryo-sections (
The analysis of the dissolution process of MNs is crucial
for reproducible antigen disposition in the skin. The
dMN dissolution time can be investigated in vitro by
immersing MNs in buffer (e.g. PBS) (
). This allows the
assessment of the quantity and quality of the dissolved
antigen. When focusing on dissolution in the skin, the
optimal application time of dMN in the skin can be
determined by analyzing MN length after the
predetermined application periods (
). The dMN
dissolution in ex vivo skin typically resembles the in vivo use of
MNs. However, it is important to analyze the dissolution
also in preclinical studies and in the early clinical
development because temperature and humidity conditions
may be difficult to mimic in ex vivo conditions. Careful
preclinical evaluation does not take away the need to
study microneedle dissolution in a clinical setting. The
contribution of physiological and mechanical properties
of the skin at the application site (e.g. thickness, elasticity,
etc) to the dMN dissolution rate and antigen delivery may
be substantial and should be investigated in the future.
Besides reproducible in vivo dissolution the actual dose
delivered should be determined. Actual dose delivered
can be substantially lower than the theoretical maximal
dose since the base of the microneedle has a tendency not
to dissolve completely. This is an economical risk. In that
respect arrow-shaped microneedles having a smaller base,
could have advantages above cone-shaped needles.
Quantification of Antigen/Adjuvant Dose
In Vitro Analysis
The quantification of antigen dose in dMNs is often very
challenging and it can be done in vitro by cutting the dMNs
from the baseplate and dissolving them (
) or embedding the
dMN patch in parafilm and allow MN tips to dissolve in PBS
). Then, the antigen quantification can be performed for
example by fluorescence (
), UV-vis analysis (90) or
ELISA. The antigen amount in the dMNs can be also
determined by dissolving the entire patch (MNs and back-plate)
and calculate the volume of the needles based on the needle
dimensions. In this case, a prior analysis should demonstrate
homogeneous antigen distribution in the entire patch.
However, these in vitro techniques are difficult to validate.
Furthermore, when using an adjuvant, this should also be
quantified to confirm its dose, similarly to antigen.
Ex Vivo and In Vivo Analysis
The antigen dose delivered into the skin and the
reproducibility of the antigen delivery can be determined in ex vivo or in vivo
), either indirectly by measuring the remaining
antigen in the dissolved MNs or directly by measuring the
antigen in the skin. Direct quantification can be performed by
using either radioactivity (
) or infrared imaging.
IMMUNOGENICITY OF ANTIGENS
ADMINISTERED BY DMNS: PRECLINICAL
The first successful vaccination with dissolving microneedles was
reported in 2010 (
). Table III gives a summary of the reported
immunization studies. Depending on the antigen, a humoral
and/or cellular response is important for a therapeutic effect.
Animal Models and Application Method
Mice are the most frequently used animal model, particularly
B a l B / c (
2 3 , 2 5 – 2 7 , 2 9 , 3 2 , 3 6 , 3 8 , 4 0
) o r C 5 7 B L / 6
) strains. Transgenic T-cell receptor mouse
models (e.g. OT-I mouse for examining CD8+ T-cell response)
can be also used as immunological model (
animal models with skin anatomy that mimics more closely
human skin may be more relevant, for example guinea pigs
for influenza (39), beagle dogs for rabies vaccination (
rhesus macaques for measles and polio vaccination (
The dMN patch can be applied either manually,
particularly if MN length is over 500 μm, (
by using an applicator (
). The advantages of
the manual application are simple administration and
reduced costs (30). However, efficient skin piercing after manual
application might be limited to longer MNs (>550 μm) while
shorter MNs (300 μm) might require an applicator (
Besides the penetration efficiency, an applicator improves
the reproducibility of the piercing, that is expected to lead to
a more reproducible delivery of the vaccine (97).
Humoral Immune Response
The model antigen ovalbumin (OVA) is most commonly used
in dMN immunization studies due to its relatively low cost and
excellent stability (
) and the strong immunogenicity in mice.
However, these beneficial characteristics mean that the results
obtained with OVA may obscure formulation problems with
more relevant vaccine antigens. Several studies with
OVAcontaining dMNs have shown that IgG responses are either
equal or superior to the ones obtained by s.c., i.m. or
traditional i.d. injection of the same dose (
Furthermore, non adjuvanted OVA dMNs (10 μg) showed a
higher response than topical application of cholera
toxinadjuvanted OVA (100 μg) on intact skin (
). This indicates
the importance of a direct delivery of the entire antigen dose
into the skin to induce an immune response.
In another study, OVA loaded chitosan dMNs elicited
higher IgG response than i.m. injection of OVA solution after
single immunization in rats. This can be explained by a gradual
degradation of chitosan microneedles creating a depot effect in
the skin (
). The OVA containing chitosan microneedles were
mounted on a PLA support. After application, the chitosan
microneedle tips were released from the support, forming a
depot in the skin. Even two weeks after the dMN application,
chitosan and OVA were still present in rat skin. Similarly, single
immunization with cross-linked silk/poly(acrylic acid) (PAA)
dMNs evoked higher IgG response than the i.d. injection of
). However, in this case sustained release from the
cross-linked silk in the PAA dMNs (100% within 12 days) did
not improve the response compared to fast release from PAA
dMNs (100% within 6 days). (
). Similarly, single
immunization with Quil-A adjuvanted OVA dMNs resulted in stronger
long-lasting IgG response than Quil-A adjuvanted OVA after
i.m immunization (
). Twenty-eight days after a single
immunization, dMNs (dose 7.6 μg) had similar IgG response to i.m
injection (15 μg) despite the lower dose. At day 102, the IgG
response of dMNs (7.6 μg) was higher than that of i.m (15 μg),
and even more interestingly low-dose dMNs (0.4 μg) had
similar response to i.m. immunization (15 μg) (
). However, it
must be noted that dMN patches were applied at two sites (both
ears) while i.m injection was performed only at one site.
Draining to two lymph nodes may have an effect on the
2 times after
2 times after
3 times after
3 times after
2 times after
Upper back skin
2 times after
VNT titers equivalent to (
that of s.c. group
IgG comparable to i.m. group (
VNT titers comparable
(42 days after the prime) and
higher (56 days after the
prime) than i.m. group
VNT titers lower than i.m.
Inner ear pinna
Ad adenovirus, B Brisbane, CpG ODN CpG oligodeoxynucleotides, DT diphtheria toxoid, EV71 Enterovirus 71, HA hemagglutinin, HBV hepatitis B virus, HIV
human immunodeficiency virus, HN hemagglutinin and neuraminidase, IPV inactivated polio vaccine, MPLA monophosphoryl lipid A, NPs nanoparticles, OVA
ovalbumin, PLGA poly-D,L-lactide-co-glycolide, poly(I:C) polyinosinic-polycytidylic acid, TIV trivalent influenza vaccine, TT tetanus toxoid, VLP virus like particles,
VNT virus neutralization test, VP virus particles
magnitude of the response. Also, the ear is a very sensitive
location for dermal vaccination probably for the short distance
to one major draining lymph node (
The use of dMNs have been shown to affect the Th1/Th2
balance. Single immunization with cross-linked silk/PAA
dMNs evoked strong IgG1 and IgG2c response while i.d.
injection elicit only IgG1 response, and thus dMN
immunization shifted Th1/Th2 balance toward Th1 (
). These results
were supported by another study where hyaluronan-based
OVA dMNs were compared to s.c. and i.d. injections in mice
). In contrast, in rats no IgG2c response was detected
neither after dMN, s.c., or i.d. immunization in the same study
). Additionally, in another study the shift in Th1/Th2
balance was not observed after dMN immunization in mice (
As conclusion, dMN vaccination may affect the Th1/Th2
balance but further studies are needed since the number of
publications on this subject is limited.
Immunization with influenza vaccine loaded dMNs resulted
often in higher ((
)) or comparable (
) IgG response than
i.m. administration. However, Kommareddy et al. showed that
dMNs evoked lower IgG response than i.m. immunization
after the boost, although the response induced by dMNs was
higher after the prime (
). However, in other studies
contradicting results were found. Haemagglutination
inhibition titers and antibodies and neutralizing antibody titers
after the dMN immunization were similar (
) or superior
) to i.m. immunization. Stabilization of the antigen by
addition of sucrose (
) may have allowed to obtain a higher
antibody titers than the previous work (
). Furthermore, the
difference with the above mentioned study (
) could be
explained by the use of a different assay (ELISA assay) than the
one routinely used to investigate the influenza vaccine quality
(single radial immunodiffusion (SRID) assay). Interestingly, the
dry matrix of dMNs can stabilize the antigen up to one year in
comparison to liquid formulation (
). In summary, most
studies show that influenza vaccination by dMNs can evoke
comparable or even superior responses than i.m. immunization.
Different types of antigen, such as vector, live attenuated and
inactivated vaccines, have been loaded in dMNs and
evaluated in vivo. An example is the vaccination of rats with the model
antigen adenovirus (Ad) loaded dMNs: Ad-specific IgG titers
observed were comparable to the s.c. group, while topical
application showed no IgG response (
). In a study
examining the dose-sparing effect, mice were immunized with dMNs
loaded with 1/10th the dose of Enterovirus71 (EV71) –
viruslike particles compared to immunization with a full dose i.m.
and s.c. injected vaccine. Antibody and neutralizing titers both
revealed comparable responses to i.m. and higher responses
than s.c. after the three immunizations. Furthermore, the
dMN group, together with s.c. and i.m. groups, survived the
lethal virus challenge showing the protective effect of the
). Rhesus macaques were used as animal model to
examine the immune response after vaccination with
inactivated polio vaccine (IPV) (
) and live-attenuated
measles vaccine (
). In both cases, neutralizing antibody titers
after dMN immunization were comparable to that after s.c.
(measles) and i.m. (IPV) immunization.
In the case of dMNs loaded with DNA containing the
rabies G-protein gene, comparable neutralizing antibody titers
with i.m. were detected after a booster. No evidence of the
dose sparing in dMNs was found since the antibody titers of
10-fold lower dose were clearly weaker than those of full dose
in dMNs (
). The co-encapsulation of plasmid vector against
hepatitis B virus (HBV) and CpG in cationic liposomes in
dMNs resulted in slightly higher IgG titers than free antigen
and adjuvant in dMNs (
). It should be considered that the
characteristics of liposomes changed after loading in dMNs
(increase in size and decrease in Z-potential). However, the
immune responses were generally similar between dMN and
i.m immunization, and adjuvant and liposomes did not affect
the IgG1/IgG2a balance (
Cellular Immune Response
De Muth et al. have reported two studies in which dMN
immunization elicited high CD8+ T-cell responses. Mice were
immunized with dMNs made of fast-dissolving PAA
containing OVA mixed with PLGA microparticles (size 1.6 μm)
encapsulating poly(I:C) (
) or cross-linked silk structure of OVA
and poly(I:C) (
). The latter results in a binary release profile:
a burst of OVA after dMN dissolution followed by a sustained
OVA release from the cross-linked silk structure. Both studies
indicated that the CD8+ T-cells producing IFN-γ and
TNF-α, upon peptide stimulation, are increased by dermal
sustained release (>16 days) from dMNs in comparison with
i.m. injection of sustained release of poly(I:C) from PLGA
), or with i.d. injection of soluble OVA and
). Furthermore, when comparing the different
dMNs, the sustained release of cross-linked silk/PAA
microneedles additionally increased the CD8+ response in
comparison with fast release of PAA microneedles (
addition, a prime immunization with dMNs can produce a
similar fraction of functional CD8+ T-cells as a prime and
boost with i.d. injection (
). Despite a larger effector CD8+
T-cell response, dMN delivery also resulted in a more rapid
transition to central memory CD8+ T-cells than i.m. and i.d.
injections, suggesting the additional expansion of CD8+
Tcells after dMN delivery did not solely result in more
terminally differentiated effector cells (
). However, sustained
release from dMNs did not further improve the memory
). A long-term memory immune response was
reported also after vaccination by Na-CMC dMNs loading
recombinant adenovirus vector encoding HIV-1 gag.
Vaccination by dMNs generated CD8+ memory T-cells
comparable with the intradermal injection (
results have been found also with other MN technologies
inducing a better long-term memory response than s.c. (
) or i.d.
The PLGA NPs dMN concept may have potential for
therapeutic cancer vaccination: dMN immunization
suppressed the growth of melanoma tumor, evoked in mice by
injecting OVA-tumor cells, through antigen-specific CD8+ T
). Furthermore, OVA-PLGA NPs dMN
immunization protected against respiratory viral challenge with a
recombinant Sendai virus expressing OVA (
). The vaccine
depot and particulate vaccines may induce a better T-cell
immune protection because the response correlates with
antigen persistence (
), the sustained antigen release (
particulate nature of vaccine. To elucidate the immunological
mechanism, it was shown that Langerhans cells are required
for cytotoxic CD8+ responses (
). Langerhans cells
apparently efficiently process the OVA loaded in the
microparticles which leads to cross presentation by MHC class I
molecules. To support this explanation, the role of
Langerhans cells was less significant for soluble OVA
compared to particulate OVA (
In another study with dMNs loaded with EV71 virus-like
particles, vaccination by dMNs loading 10 times lower antigen
dose than i.m. and s.c. injections could promote stronger
EV71specific T-cell response than the conventional injections (
Viral vectors are able to induce strong T-cell responses
after dMN immunization. dMNs with human adenovirus
expressing ovalbumin were compared to i.d, i.m. and s.c.
injections. The T-cell responses were similar in all groups (
Similarly, CD8+ T-cell responses were comparable after mice
were immunized with rAdHu5 vector encoding a HIV-1 Gag
gene by dMNs or i.d. injection (
dMN Immunization: Factors Influencing
Several adjuvants have been used in dMNs and they are
similar to those used for other administration routes except
aluminum based adjuvants and emulsions. Aluminium based
adjuvants may cause local adverse effects like granuloma
formation and therefore is not suitable for delivery to the skin (
Emulsions cannot be formulated in dry formulations like
dMN because water is a structural part of the formulation.
Molecular immune modulators, such as CpG (
poly(I:C) (34), Quil-A (
), monophosphoryl lipid A (MPLA)
) and imiquimod (
) have been used in dMNs. In
general, a significant increase in the immune response is observed
when an adjuvant is included in dMNs (
sometimes the control group without the adjuvant is lacking.
Unfortunately, the rationale of selection a certain adjuvant
and its dose has not often been addressed.
Delivery systems can be formulated into dMNs (see
previous section). Similarly to other administration routes,
encapsulation of antigen (and adjuvant) in nanoparticles or liposomes
can enhance the immune response after delivery with dMNs
) as described above (Humoral Immune Response
and Cellular Immune Response Sections). Adjuvants are often
needed with modern subunit vaccines but their use might be
avoided with attenuated viruses and viral vectors. Absence of
adjuvant would also facilitate batch release since adjuvant
quantification is not needed and antigen quantity is often
limited to a simple plaque titration or colony count as opposed to
an immunogenicity test in experimental animals.
MNs Spacing and MN Geometry
Modelling studies have indicated that the MNs spacing may
affect the immune response by contributing to the optimal
antigen concentration released into the skin to activate APC
located between the MNs (
). However, this is not
experimentally confirmed and factors not accounted for in the
model may contribute significantly to the immunogenicity.
The MN length may influence the population of APCs
activated so that shorter MNs could activate LCs in the
epidermis and longer MNs could activate dDCs in the dermis
). In vivo studies with 1 μg OVA showed that IgG response
after vaccination by dMNs of 300 and 800 μm in length is
higher than using dMNs of 200 μm in length (
). On the other
hand, the variation of injection depth with hollow MNs did not
affect the immune response (
). However, while a controlled
antigen dose was released at different skin depth by hollow
), not clear is the antigen dose released into
the skin from the dMN of different lengths (
). This could
explain the difference in the immune response.
Apart from MN length, needle density may be an
important variable with respect to immunogenicity. The needles
cause minor damage and cell death, initiating a pathway
acting as Bnatural immune enhancer^ mediated by the release of
damage-associated molecular patterns (DAMPs) (
). In fact,
the same antigen dose released by coated MNs elicited higher
response than a single i.d. injection (
CLINICAL DEVELOPMENT OF DISSOLVING
dMNs are a relatively new vaccine delivery system with no
licensed vaccines and few results from clinical studies. Two
phase 1 (safety) studies with microneedles without antigen
have been performed so far. In the first, hyaluronan
microneedles (length 300, 500 and 800 μm, 200 dMNs
on a 0.8 cm2 patch) have been applied on 17 subjects
). Despite a successful dMN penetration into the skin
by using an applicator, the microneedles required 6 h of
application for nearly complete dissolution in all subjects,
which may be too long for routine immunization. In the
second study, PVA microneedles (length 650 μm, 100
dMNs on a 1 cm2 patch) have been applied on 15 subjects
). In this case, an average of 100% piercing efficiency
of MNs into the skin without any applicator use was
reached. However, variance in the microneedle volume
dissolved, especially among subjects using
self-administration, underlined the importance of using an applicator to
have a controlled force and an impact during application.
Few subjects (
) or all of them (
) showed a slight
erythema after dMN application that disappeared within
7 days. However, longer dMNs of 500 and 800 μm caused
purpura, indicating capillary damage, in 50% of the
volunteers but shorter 300 μm dMNs did not induce any
). No swelling at the application site (
systemic adverse events were observed (
). Additionally, it
was also concluded that dMN application caused hardly
) or no pain (
In another clinical phase 1 study, trivalent influenza
hemagglutinins vaccination with sodium hyaluronate dMNs
(800 μm, 200 dMNs on a 0.8 cm2 patch, spring-type
applicator used) was investigated in healthy subjects (
loaded with 3 x 15 μg of influenza antigens on a single patch,
were compared with the same dose administered by s.c.
injection. During the prime immunization a proper dMN
dissolution was observed in only seven subjects out of 20 and only
these subjects were included in the final analysis. Furthermore,
the applicator settings were changed to obtained a more
efficient application in the second vaccination. After the prime,
the anti-HI antibody titers against influenza A HA-antigens
(H1N1 and H3N2 strains) were equivalent in the dMN and
s.c. groups, except that for the B strain that showed higher
titers in the dMN group also observed in preclinical studies
). More IFN-γ-producing peripheral blood mononuclear
cells were detected after s.c. than dMN immunization (
However, the low number of subjects in dMN group limits the
conclusions. Regarding the safety, erythema detected in the
dMN group was higher than the s.c. one and more
pronounced than in the previous clinical studies (
Purpura was observed in 50% of the subjects both in the
dMN and the s.c. group. However, no adverse systemic events
were observed (112).
These studies prove that the applicator and its settings have
a crucial role for MN penetration and subsequent dissolution
into the skin. Alternatively, encapsulation of the antigen only
in the microneedle tip can enable a complete antigen delivery
Table IV Target Product Profile of
the Ideal dMN Patch
Total systems costs lower than injected vaccine
Competitive production costs
Simple to produce
Stable outside the cold chain
Higher immunogenicity / dose sparing / single shot
No applicator needed
Fail-proof application/check on full dose delivery
Short application time
Less adverse effects
even with incomplete microneedle dissolution (e.g., localizing
the antigen in the upper 70% of the MNs, a 70% dissolution
would correspond to 100% antigen delivery).
Besides above mentioned studies, at least one other study
has been performed to investigate safety and immunogenicity
of influenza vaccination with dMNs but the results are not yet
CONCLUSIONS AND PROSPECTS
dMN vaccination can offer important advantages such as dose
sparing, pain-free immunization and avoidance of
needlestick injuries. Furthermore, it can extend the vaccination
coverage in developing countries by potentially offering improved
vaccine stability, reduction of vaccine wastage and of burden
on trained personnel. However, several improvements are still
needed in some areas of dMN development before the
regulatory acceptance and industrial scale-up are feasible.
Fabrication methods require further optimization to enable
the minimal wastage of antigen that is often claimed but rarely
reported in the literature, and not yet proven on at least pilot
scale production level. Analytical challenges include potency
testing and stability testing during fabrication and storage, and
quantification and reproducibility of antigen/adjuvant dose
delivered in the skin. Furthermore, the role of the applicator
device should not be underestimated because it can
standardize dMN application and vaccine delivery into the skin,
although with respect to logistics manual application is
preferred. dMN immunization has generated comparable or
higher and more durable antibody and cellular responses than
conventional immunizations in preclinical studies.
Additionally, sustained release of antigen from nanoparticles
or cross-linked structures in dMNs showed to induce a better
cellular immune response than fast release from dMNs or
liquid solution, although the sustained release from dMNs
did not improve further the humoral response than fast release
from dMNs. However, further studies should be performed to
support this conclusion. In the future, more systematic studies,
such as identification of optimal adjuvants and analysis of
effect of dMN geometry, may be necessary to optimize
dMN immunization. Until now, three clinical phase 1 studies
have been reported and showed that skin irritation and patch
application are hurdles that need to be solved in future
applications. The ideal dMN patch (Table IV) does not exist yet
but encouraging progress has been made. More work is
needed to further develop dMNs into safe, efficacious, affordable
and widely used products.
ACKNOWLEDGEMENTS AND DISCLOSURES
This work was sponsored by Intravacc.
Open Access This article is distributed under the terms of the
Creative Commons Attribution 4.0 International License
permits unrestricted use, distribution, and reproduction in any
medium, provided you give appropriate credit to the original
author(s) and the source, provide a link to the Creative
Commons license, and indicate if changes were made.
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