Review

Therapeutic
Delivery
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Intranasal delivery of nanoparticle-based

vaccines

Most pathogens gain access to the human body and initiate systemic infections through Nirmal Marasini1,2, Mariusz
mucosal sites. A large number of currently marketed licensed vaccines are parenterally Skwarczynski**,1 & Istvan
administered; they generate strong systemic immunity but not mucosal immunity. Toth*,1,3,4
1
School of Chemistry & Molecular
Nasal vaccination is an appealing strategy for the induction of mucosal-specific
Biosciences, The University of
immunity; however, its development is mostly challenged by several factors, such as Queensland, St Lucia, QLD, 4072,
inefficient antigen uptake, its rapid mucociliary clearance, size-restricted permeation Australia
across epithelial barriers and absence of safe human mucosal adjuvants. Therefore, 2
School of Biomedical Sciences, The
a safer mucosal-adjuvanting strategy or efficient mucosal delivery platform is much University of Queensland, St Lucia, QLD,
4072, Australia
warranted. This review summarizes challenges and the rationale for nasal vaccine 3
Institute for Molecular Biosciences, The
development with a special focus on the use of nanoparticles based on polymers and University of Queensland, St Lucia, QLD,
lipids for mucosal vaccine delivery. 4072, Australia
4
School of Pharmacy, The University of
First draft submitted: 28 September 2016; Accepted for publication: 18 November Queensland, Woolloongabba, QLD 4102,
Australia
2016; Published online: 31 January 2017
**Author for correspondence:
m.skwarczynski@ uq.edu.au
Keywords: lipids • mucosal immunity • nanoparticles • nasal vaccines • polymers *Author for correspondence:
Tel.: +61 7 3346 9892
Fax: +61 7 3365 4273
Since the introduction of the first vaccine mucosal routes such as oral, nasal, pulmo- i.toth@ uq.edu.au
in the 19th century, mass vaccination pro- nary, ocular, rectal, vaginal, etc. Among
grams have saved millions of lives. Although mucosal routes, the simplest, convenient and
the incidence of vaccine-preventable diseases preferred routes for vaccine administration
is less common in developed countries, as are the oral and intranasal. However, the
many as 15 million people are still dying oral route is the most challenging for vaccine
yearly due to infectious diseases [1] . Most administration due to antigen degradation by
pathogens gain access to the human body proteolytic enzymes and acidic pH in the GI
through mucosal sites and initiate systemic tract [2] . In contrast, the intranasal admin-
infections. Currently, marketed licensed vac- istration route offers fairly low enzymatic
cines are parenterally administered but these degradation of antigens compared with other
only generate strong systemic immunity and mucosal routes, houses a large number of
almost no mucosal immunity. The lack of immune cells, and mucosal epithelial mem-
mucosal vaccines against existing and emerg- branes are relatively easily permeable [3] . In
ing pathogens has created the need for the addition, nasal vaccines possess several advan-
development of a novel and effective muco- tages: for example, they are needle-free and
sal vaccination strategy, which can induce noninvasive in nature, offer the possibility of
both antigen-specific systemic and mucosal mass immunization and require less trained
immunity. Unfortunately, the development manpower for immunization [4] . The differ-
is partly limited due to the absence of safe ences between the intranasal and oral routes
human-approved mucosal adjuvants. of vaccine administration are summarized in
Mucosal immune responses are typically Table 1. Despite several attractive features of
part of
induced when antigens are delivered through intranasal vaccination, only a few intranasal

10.4155/tde-2016-0068 © 2017 Future Science Ltd Ther. Deliv. (2017) 8(3), 151–167 ISSN 2041-5990 151
Review Marasini, Skwarczynski & Toth
Table 1. Differences between intranasal and oral route of vaccinations.
Intranasal
Nasal mucosal surface Highly vascularized
Absorption Rapid absorption of antigens to reach
systemic circulation
First-pass metabolism (presystemic Avoids first-pass effect
metabolism)
Influence of enzymes and pH Low influence of proteolytic enzymes and
pH
Required dose Relatively lower than oral/less frequent
immunizations
Immune responses Rapid local immune responses
Protective immune responses in lungs,
upper respiratory tract and genital tract
mucosae
Poor intestinal immune response
vaccines (Flumist®, Fluenz® and Nasovac®) are avail-
able for human use [5–7] . Nasal vaccine development is
mostly challenged by the inefficient uptake of soluble
antigens by nasal immune cells, rapid mucociliary
clearance or short resident time of antigens in the nasal
tract, size-restricted permeation across epithelial bar-
riers, absence of safe human mucosal adjuvants, poor
antigen stability and restricted delivery volume.
Vaccines usually contain killed or live-attenuated
organisms or their subunit fragments: for example, car-
bohydrate, proteins and peptides. Historically, whole
pathogen-based vaccines were the most popular, mostly
because of the simplicity and relatively low cost of their
production. The success of such vaccines was also related
to their capability of presentation of multiple antigens,
along with natural adjuvants, to the immune system that
ultimately led to a potent antibody and cell-mediated
immunity. However, the use of whole pathogen-based
vaccines comes with some demerits such as chances
of autoimmune or strong allergic responses, produc-
tion difficulties (not all pathogens can be cultured),
biological contamination, etc. [8] . Therefore, alterna-
tive methods using subunit antigens such as toxins or
small-pathogen fragments (proteins, carbohydrates
and peptides) provide a rational approach toward safe
immunizations. Unfortunately, induction of the desired
immune responses with subunit-based antigens is often
difficult to achieve even after parenteral immunization,
as they are poorly immunogenic due to the lack of dan-
ger signals. Therefore, an adjuvant or delivery system is
required to enhance the immunogenic efficacy of sub-
unit vaccines. Recently, nanoparticles have been pro-
posed as a promising self-adjuvanting delivery system to
overcome the lack of effective mucosal adjuvant.
152 Ther. Deliv. (2017) 8(3)
Oral
Less vascularized than nasal mucosal
surface
Inconsistent absorption profile/longer time
to reach systemic circulation
Extensive first-pass metabolism
High influence of enzymes and varying pH
across GI tract
High-dose/more frequent immunization
Slower or unresponsive to soluble antigens
Protective immune responses at GI tract,
upper respiratory tract (tonsils), salivary
and mammary glands
Nasal immune system
Nasal mucosa is an important part of the immune sys-
tem since it is a first site of contact for inhaled patho-
gens or vaccine-associated antigens. The nasal mucosa
consists of epithelial cells, which play a crucial role in
maintaining immune homeostasis by providing a con-
tinuous physical barrier against exogenous substances.
The predominant immunoglobulin found in nasal
mucosa is secretory IgA (sIgA). High levels of local
mucosal antibodies are usually produced at the site of
antigen administration and comparatively low IgA is
produced at distant mucosal sites.
The primary site for the induction of nasal immu-
nity against administered antigens is the nasal-associ-
ated lymphoid tissues (NALT). In rodents, NALT con-
tain aggregates of organized lymphoid follicles located
at the nasopharyngeal cavity [9] . This structure is func-
tionally similar to human’s Waldeyer’s ring (tonsils
and adenoids) and primarily responsible for producing
local immune reactions upon intranasal vaccination in
the rodents [10] . Therefore, the rodent-based models are
well suited for early vaccine development. NALT com-
prises B cells, T cells, macrophages and dendritic cells
(DCs)-enriched areas, which are overlayed by epithe-
lial cells, and a small number of specialized microfold
cells (M cells) (Figure 1) . M cells are primarily respon-
sible for transporting antigens from the apical side of
the lumen to DCs or B cells [11] . The particulate-based
antigens are actively transported by M cells, whereas
soluble antigens are passively absorbed from the apical
side of the epithelial membrane toward the basolateral
region. Following antigens’ transport via M cells or
epithelial barriers, resident DCs capture and process
antigens and present them locally to adjacent T cells
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 Intranasal delivery of nanoparticle-based vaccines Review

in the subepithelial region. Alternatively, subepithelial
DCs may extend their dendrites from transepithelial
junctions to capture antigens in the luminal region.
This special antigen capture behavior by DCs is exclu-
sive to the areas in the nasal mucosa where there is an
absence of organized lymphoid follicles and M cells.
Once antigens are captured by DCs’ extensions, they
are actively transported to the nearest draining lymph
nodes for antigen presentation to the CD4 + T cells [12] .
In the lymph nodes or organized lymphoid follicles,
DCs-stimulated CD4 + T cells activate naive B cells
and these B cells undergo isotype class switching to
form antigen-specific IgA+ committed B cells. These
committed IgA+ secreting B cells express high levels of
CCR10 (mucosal homing receptors) and migrate from
lymph nodes to the blood circulation. The circulating
IgA+ B cells exit the blood and the expressed CCR10
in IgA+ B cells eventually guide their entry to effector
sites in the nasal epithelial cells that express CCL28,
a mucosa-associated epithelial chemokine [13] . Finally,
IgA+ B cells undergo final differentiation and matura-
tion to generate IgA+ plasma cells (process enhanced
by cytokines such as IL-5 and IL-6, a subset of Th2
cells) and finally produce dimeric (or polymeric) forms
of IgA. The dimeric IgA binds to polymeric Ig recep-
tors (expressed by the basolateral surface of nasal epi-
thelial cells) to form sIgA. sIgA is then further trans-
cytosed toward the apical luminal surface in the nasal
passage. sIgA antibodies are able to recognize, bind or
neutralize the toxins or pathogenic micro-organisms,
thus preventing their access to internal organs and
forming the first line of defense against pathogens.
Unlike other antibodies, sIgA antibodies are special
and unique in nature as they are resistant to protease
degradation at the mucosal surface [12] . They remove
antigens by blocking access to the epithelial receptor,

Effector site pIgR G

H

sIg
gA
sIgA Dimeric IgA

Nanoparticles
ticles
F
C
Dendritic
D
Deendritic
d i i cells
ce
ells
ll

IgA+ plasma cells

M cells CD4
4+ T cell
cells
ls
s
A

IgA+ B cells
D

B
E
Mucosa

IgA+ B cells
Nasal epithelium

CD4+ T cellsh nod

p
Lym
Systemic circulation
Lymph node

Figure 1. Mechanism of immune induction in nasal-associated lymphoid tissues. The particulate antigens are
actively taken up by M cells (A) or permeate passively through epithelial junctions (B). Alternatively, antigens
are taken up by DCs extensions into the lumen (C). Once antigens are captured by DCs, they migrate to nearest
lymphoid follicles or lymph node to present antigens to CD4 + T cells. DCs-stimulated CD4 + T cells activate naive
B cells and undergo isotype switching to form antigen-specific IgA + committed B cells (D). These IgA + B cells
migrate from lymph node to the blood circulations (E). Finally, IgA + B cells exit the blood and enter toward the
effector site. They undergo final differentiation and maturations producing IgA + plasma cells (process enhanced
by IL-5 and IL-6, a subset of Th2 cells) and ultimately form dimeric or polymeric IgA (F). The dimeric or polymeric
IgA binds with pIgR in the basolateral region to form sIgA (G) and further transcytosed toward the apical side of
luminal surface (H).
DC: Dendritic cell; IL: Interleukin; M-cell: Microfold cell; pIgR: Polymeric immunoglobulin receptor; sIgA: Secretory
IgA; Th: T-helper cell.

future science group www.future-science.com 153

Review Marasini, Skwarczynski & Toth
trapping them in mucus and preventing their adher-
ence to mucus by mucociliary-mediated exclusions [13] .
Apart from mucosal sIgA, nasal immunization can
also induce systemic antibodies (IgG) in the periph-
eral lymphoid tissues as observed in parenteral vaccines
that can clear pathogens from the blood circulation.
This feature makes the intranasal route highly efficient
for the delivery of vaccines.
Nanoparticles as a mucosal adjuvanting
system
Adjuvants are usually used in modern vaccine formu-
lations to enhance the immunogenicity of the deliv-
ered antigens [14] . A significant progress in the devel-
opment of adjuvants for parenteral vaccine delivery
has been achieved in the last few decades. The most
popular adjuvant used in human vaccine formulations
is alum, due to its favorable safety profile. Although
alum triggers the induction of an antigen-specific sys-
temic immune response, it does not induce cellular and
mucosal responses [15] . Cholera toxins and heat-labile
toxin (LT) are highly effective mucosal adjuvants.
However, their use in humans has been constrained by
their toxicity and the possibility of antigens redirecting
to olfactory tissues or the CNS due to the capacity of
B subunits of cholera toxin to bind with ganglioside-
receptors presenting on all nucleated cells [16,17] . These
constraints have led to an interest in the development
of a safer and nontoxic alternative adjuvanting strategy
for nasally administered vaccines.
The application of nanoparticles in mucosal vac-
cine delivery has the potential to overcome obstacles
associated with the classical adjuvants [8] . Vaccines
formulated into nano- or micro-particles showed more
efficient antigen-presenting cells (APCs) internaliza-
tions, cross-presentation and B-/T-cell responses [18–
20] . In mucosal vaccination, the adjuvanting effects
of the nanoparticles are related to: improved ability
to cross mucosal barriers, enhanced uptake by APCs,
prolonged availability to interact with APCs and the
provision of additional danger signals due to their size
mimicry of pathogens. This mode of delivery system is
becoming increasingly popular owing to their tunable
physicochemical characteristics, capacity to protect the
native structural integrity of the antigens, presentation
of antigens into multimeric form and delivery of anti-
gens to the targeted sites, particularly the lymph node.
The limited permeation and rapid exclusion of
soluble antigens from the mucosal passage are often
the primary bottlenecks for the nasal delivery of vac-
cines. Thus, the solubility or hydrophilicity of anti-
gens (e.g., peptides or proteins) can be altered by
conjugation with lipids or carbohydrates. Addition-
ally, these modifications of antigens were shown to
154 Ther. Deliv. (2017) 8(3)
better enhance the immunogenicity of antigens com-
pared with their soluble antigen counterparts due to
the increased stability and lipophilicity of the vaccine
compounds [21–23] . However, the optimum number of
lipids or carbohydrate moieties required to enhance
the appropriate immune responses are often difficult
to predict. It was observed that increasing the length of
lipid moieties conjugated to peptide epitopes allowed
them to self-assemble into nanoparticles (Figure 2A)
and the systemic and mucosal immune responses
were increased upon intranasal immunization [24–26] .
However, the involvement of complex synthetic and
conjugation chemistries often gives a low yield, thus
limiting their mass production. Alternatively, vaccines
can be encapsulated, adsorbed, simply conjugated or
physically mixed with biomaterials such as polymers
and lipid-based nano-/microparticles (Figure 2B–F) . A
large number of synthetic/natural polymers and lipids
are widely used in intranasal drug or vaccine delivery.
Codelivery of vaccine components into a single par-
ticulate system mimics the characteristics of natural
pathogens and preserves the native structure of vac-
cines in the extracellular and intracellular fluids. The
particulate nature of polymers or lipids, depending
upon their physicochemical properties (e.g., hydrophi-
licity/lipophilicity, charge, morphology, composition,
etc.) allows them to be selectively taken up by M cells
or nonselectively by epithelial cells before reaching the
lymph nodes and additionally, to control the release of
antigens over the period of time.
Polymer-based nanoparticles for nasal
vaccination
Natural and synthetic polymers are one of the most
well-established carriers of proteins, peptides and DNA-
based vaccines. The most commonly used p­olymers in
nasal vaccine delivery are polyesters derivatives such
as poly(lactic-co-glycolic acid; PLGA), polylactic acid
(PLA) and their copolymers, polysaccharides (such
as chitosan, alginate and dextran), poly(amino acids)
[such as poly(γ-glutamic acid) (γ-PGA), poly( l -lysine),
poly( l -arginine)] and polyacrylates.
Polyesters
PLGA and PLA are the best examples of polyester
polymers with safe, biodegradable and biocompat-
ible properties. The well-documented safety history
of PLGA has resulted in a number of applications for
drug delivery. The polymer has been examined in sev-
eral clinical trials and marketed in drug formulations
but has yet to be successfully applied in commercial
vaccines [27,28] . Both PLGA and PLA nanoparticles are
usually prepared by solvent-evaporation/emulsification
and nanoprecipitation methods that allow isolating
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 Intranasal delivery of nanoparticle-based vaccines Review

A B C

D E F

Figure 2. Different methods of antigen incorporation to nanoparticles. (A) Amphiphilic antigens can self-
assembled itself to form nanoparticles, (B) antigens are encapsulated into the nanoparticles, (C) antigens are
adsorbed on the surface and encapsulated into the nanoparticles, (D) antigens are adsorbed on the surface
of nanoparticles, (E) antigens are conjugated onto the surface of the nanoparticles and (F) physical mixture of
antigens and nanoparticles.
particles of ultrasmall size up to a minimum of 70 and due to poor mucoadhesiveness and immune-activating
20 nm, respectively [29,30] . Both the particle prepara- properties [33,34] . It was previously shown that positive-
tion processes require antigens or polymers to be dis- charged particles are more favored for enhanced muco-
solved into organic solvents that may cause denatur- adhesiveness over negative-charged particles [33,35,36] .
ation of antigens (especially proteins). Additionally, The carboxylic group (–COOH) of PLGA imparts a
these techniques often require the use of surfactants to negative charge to PLGA that disfavors interaction of
stabilize the particles. Therefore, the major difficulty PLGA with the mucosal surface leading to rapid expul-
during the production is to remove extra traces of the sions of particles from the nasal mucosal surface, thus
remaining surfactants in order to avoid cytotoxic side making fewer particles available for activation of the
affects [28] . immune system. Slütter et al. demonstrated that the
Polyesters polymers are able to encapsulate/adsorb in vivo nasal residence time of PLGA nanoparticles
large amounts of both hydrophilic and lipophilic anti- encapsulating ovalbumin (OVA) protein as antigens
gens and immunomodulatory compounds as a cargo was similar to the soluble OVA protein [37] . Polyesters
into the core/surface of a single particulate system [31] . were coated with polyethylene glycol (PEG) or polox-
These polymers are eroded slowly by the hydrolysis of amer 188 to increase hydrophilicity, circulation time
ester linkages into nontoxic metabolites (e.g., lactic and biological stability of the nanoparticles [38,39] .
acid and glycolic acids for PLGA) before their elimi- Vila et al. prepared PLA nanoparticles with or without
nation by the kidney [32] . While PLGA is extensively PEG-encapsulating tetanus toxoid (TT) antigens to
used parenterally, its use in nasal vaccination is limited compare their biological stability and i­mmunological

future science group www.future-science.com 155

Review Marasini, Skwarczynski & Toth
fate upon intranasal administration. While PLA
nanoparticles without PEG aggregated in the pres-
ence of lysozyme, PEG-coated PLA nanoparticles were
stable [39] . Furthermore, in vivo evaluation of both
the nanoparticles showed that PEG–PLA nanopar-
ticles penetrated nasal mucosae efficiently and elicited
higher and more long-lasting immune responses com-
pared with PLA-only nanoparticles. Cu et al. showed
that PEG coating of the PLGA nanoparticles improved
particle diffusion across the mucosal barrier up to ten-
fold in comparison to uncoated particles [40] . Thus,
PEG coating on the surface of polyesters may provide
an effective approach to deliver antigens by enhancing
their stability and in vivo efficiency.
The low efficacy of vaccines due to reduced muco-
sal permeation of antigen can be overcome by spe-
cifically targeting nanoparticles to nasal M cells
that may allow the rapid transport of nanoparticles
to the underlying lymphoid tissues. There are only
a few reports on the use of M-cell-targeting ligands
employed with polyester-based nanoparticles for
nasal vaccine delivery. Rajapaksa et al. encapsulated
fusion protein into PLGA nanoparticles incorporat-
ing a mixture of protein antigens hemagglutinin-
histidine tagged (HA–HT) protein antigens and a
C-terminal targeting peptide derived from Clostrid-
ium perfringens enterotoxin (CPE) to bind Claudin
4 receptors, which are expressed in both human and
mouse M cells [41] . Nanoparticles prepared with tar-
geting peptide were taken up significantly better by
NALT M cells compared with nontargeted nanopar-
ticles. The same group also reported the influence of
dispersion of PLGA nanoparticles formulated with or
without M cells targeting ligand or poloxamer 188
at various ionic strengths of buffers on antigen in
vivo uptake by NALT M cells following intranasal
immunizations [38] . The nanoparticles were dispersed
into buffers with different ionic strengths ranging
from as high as 1.5 × 10 -1 M to a low ionic strength
1 × 10 -7 M at pH 7, while osmolarity was controlled
by addition of mannitol to bypass any influence of
osmolarity on uptake. Upon intranasal immuniza-
tion, nanoparticles were highly taken up by NALT
at low ionic strength of phosphate buffer regardless
of the presence of targeting peptides or poloxamer.
Thus, the capacity of long-term electrostatic interac-
tions between the particles and mucosa (mucoadhe-
siveness) was the more important factor influencing
antigen uptake in NALT than the use of a targeting
moiety. Therefore, several efforts were undertaken to
modify the surface of PLGA nanoparticles by using
mucoadhesive polymers (such as chitosan and their
derivatives) to improve their mucus-binding efficacy
and nasal residence time [35,37,42] .
156 Ther. Deliv. (2017) 8(3)
Chitosan & their derivatives
Chitosan is linear polysaccharide predominantly com-
posed of β-(1–4)-linked d -glucosamine and N-ace-
tyl- d -glucosamine. The N-acetyl- d -glucosamine moi-
ety of chitosan is recognized by the mannose receptors
on the DCs [43] . Chitosan is produced by deacetylation
of naturally occurring chitin (component of exoskel-
etons obtained from crustaceans such as shrimps and
crabs) using a hot alkaline solution (aqueous sodium
hydroxides) [44] . Chitosan is biodegradable and showed
an excellent safety profile with low toxicity [45] . Due to
their mucoadhesive and immunostimulating proper-
ties, chitosan is a promising alternative to poly(esters)
for a nasal vaccine carrier [46] . The enhanced mucoad-
hesive property of chitosan is mainly attributed to the
presence of the polycationic charge (amine moieties)
in the chitosan that binds with anionic sialic acids (a
principle component in mucus) [47] . Additionally, chi-
tosan promotes the opening of the tight junctions in
the epithelial membrane for efficient permeation of
particles [48] . However, recent study showed that such
opening of tight epithelial junctions in the presence of
chitosan might be not efficient for antigen transporta-
tion as it did not help ovalbumin protein to cross the
epithelial barrier [49] .
Vila et al. used chitosan nanoparticles for the intra-
nasal delivery of TT as model antigens in mice [50] .
High- and long-lasting antigen-specific serum IgG
and mucosal IgA antibody titers were detected upon
immunization with chitosan nanoparticles. The effi-
ciency of chitosan nanoparticles in generating humoral
immune responses was independent of the molecular
weight of the chitosan. Chitosan nanoparticles have
also been successfully applied as a nonviral carrier
for DNA vaccines [51,52] . Lebre et al. used chitosan
nanoparticles, human serum albumin and plasmid
DNA (encoding hepatitis B surface antigens [HBsAg])
to develop a vaccine against the hepatitis B virus [52] .
The nanoparticles were able to show high transfection
efficiency and induce significant humoral and mucosal
responses against hepatitis B virus.
Although chitosan nanoparticles possess inherent
adjuvant properties, they are also used in conjunc-
tion with other immunomodulatory compounds
such as CpG oligodeoxynucleotide, Quillaja saponins
(QS), monophosphoryl lipid A and mast cell-activat-
ing adjuvant compound 48/80 (C48/80) for nasal
i­mmunizations [53–56] .
The amine group of chitosan has a dissociation con-
stant (pK a) 6.2, thus at pH > 6.2 chitosan is deprot-
onated, making them insoluble in water. This could
prevent encapsulation of antigens that are only soluble
at a neutral or higher pH. Therefore, chitosan is often
modified to improve its aqueous solubility in a wider
future science group

range of pH [57] . For example, the chitosan amine
group can be methylated to form trimethyl chitosan
(TMC), which bears a permanently ionized quater-
nary amine group [58–60] . The TMC nanoparticles can
be simply prepared at ambient temperature by ionic
gelation of cationic TMC and anionic tripolyphos-
phate under stirring, without the requirement of soni-
cation and presence of organic solvents, thus providing
a lower possibility of antigen denaturation [59] .
The mode of antigen delivery could play an impor-
tant role in determining the immunogenicity of anti-
gens. Antigens can be delivered with chitosan or its
derivatives as physical mixtures or as a conjugate with
antigen [61–64] . Covalent conjugation of an antigen
with chitosan-based polymers shows better systemic
and mucosal antibody responses than antigens encap-
sulated or mixed with chitosan [61–63] . The conjugate
approach is most beneficial for the delivery of low
immunogenic protein and peptide antigens.
Chitosan has also been used to modify the sur-
face properties of anionic polyesters. Coating anionic
PLGA particle surfaces with chitosan-based polymers
led to the formation of the cationic nanoparticles with
slightly increased size, improved nasal residence and
enhanced humoral immunity against administered
antigens [35,37] . Taken together, in contrast to the
majority of classical adjuvants which are associated
with some degree of side-effects or toxicity, the use of
highly immunogenic chitosan-based nanoparticles is a
promising intranasal vaccine delivery strategy.
Alginates
Alginate is a hydrophilic anionic polysaccharide
obtained from brown algae and mostly used in drug
and vaccine delivery [2,65] . It is nontoxic, biodegradable
and biocompatible. Alginates can be used to encapsu-
late an antigen, protect it from enzymatic degradation
and facilitate its suitable release. Although the appli-
cation of alginate as a carrier in an intranasal vaccine
delivery system was reported, the polymer is mainly
used for the oral delivery of antigens.
Borges et al. demonstrated that alginate coating of
the HBsAg/chitosan complex induced a detectable
mucosal response in nasal washes but did not pro-
duce sufficient systemic responses upon intranasal
immunization [66] . The authors suggested that the
poor immune responses might be related to muco-
sally induced tolerance against the antigens. Addition-
ally, the low mucoadhesive property of alginate could
explain the poor efficacy of nanoparticles. In a similar
study, antigen encapsulated into chitosan and chito-
san/alginate nanoparticles was compared for nasal
immunization [67] . In contrast to chitosan-only bear-
ing nanoparticles, chitosan/alginate nanoparticles
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Intranasal delivery of nanoparticle-based vaccines Review
were less efficiently taken up by APCs, showed low
nasal residence time and induced weak systemic and
mucosal antibody titers. This further signifies that
incorporation of alginate into the delivery system neg-
atively affects vaccine responses, indicating that algi-
nate in an unfavorable polymer for intranasal delivery
of vaccines.
Dextran
Dextran is a polysaccharide consisting of a linear
repeating unit of α-1, 6-linked glucopyranose. It is
used in drug delivery as mucosal absorption enhanc-
ers. Dextran is efficiently recognized by the mannose
receptor present on the DCs [68] .
Dextran is a hydrophilic polymer and its derivative
(dextran sodium) is most commonly used in drug or vac-
cine delivery. Dextran sulfate is polyanionic due to the
presence of a large number of sulfate groups [69] . Chen
and colleagues developed dextran/chitosan nanopar-
ticles with IgA as targeting ligand using the polyelec-
trolyte complexation/ionic gelation method [70] . These
nanoparticles were able to incorporate pertussis toxin
(PTX) and targeting ligand (IgA) with high entrap-
ment efficiency (>90%). Nanoparticles were easily
uptaken in vivo in the NALT area. However, no in vivo
immunization was conducted to show the efficacy of
particles in inducing antibody responses. Jaafari and
colleagues prepared cross-linked dextran microspheres
(CDM) loaded with TT. CDM loaded with TT were
able to induce potent antigen-specific serum IgG and
IgA antibody titers in rabbits after intranasal immuni-
zation [71] . Additionally, high-serum anti-TT antitoxin
titers (175-times higher than required to achieve a pro-
tective level) were induced by dextran-based micro-
spheres. Similarly, CDM physically mixed with alginate
microspheres loaded with QS and TT induced a high
mucosal and systemic antibody response while QS and
TT co-encapsulated into alginate were less effective [72] .
Despite being negatively charged, dextran can enhance
the uptake of particles in the nasal mucosa. Although
the exact mechanism is poorly understood, it could be
hypothesized that solid dextran microsphere powder
adsorbs water from the mucus layer that may shrink the
mucosa and allow the penetration of the particles across
the mucosal barrier [72] .
Poly(amino acids)
Recently, poly(amino acids) have become increasingly
popular for delivering vaccines, genes and proteins [73] .
Poly(amino acids) contain the repetition of a small
number of the same amino acid. The poly(amino acids)
can be conjugated between each other (hydrophobic and
hydrophilic poly(amino acids)) to form an amphiphi-
lic block copolymer-like structure. In an aqueous solu-
www.future-science.com 157
Review Marasini, Skwarczynski & Toth
tion, an amphiphilic poly(amino acids)-based system
can form nanoparticles by spontaneous self-assembly of
their hydrophilic and hydrophobic moieties [74] .
γ-PGA is a safe, biodegradable natural material that
was examined for the delivery of vaccines against vari-
ous diseases such as influenza, anthrax and noninva-
sive cancers [75–77] . Matsuo et al. conjugated hydropho-
bic l -phenylalanine ethyl ester ( l -PAE) into α-position
of carboxyl functional group of γ-PGA and self-assem-
bled into nanoparticles (γ-PGA- l -PAE) that effectively
entrapped tumor-associated antigen (OVA) [77] . Upon
intranasal vaccination with γ-PGA- l -PAE nanopar-
ticles, highly potent tumor-specific long-term cellular
immune responses were observed. Additionally, this
vaccine induced strong systemic IgG titers, whereas
very low mucosal IgA titers were detected. In contrast,
when influenza virus hemagglutinin (HA)-loaded
γ-PGA nanoparticles were administered nasally, they
elicited high mucosal antibody titers and cross-protec-
tive immune responses sufficient for mice survival [75] .
Interestingly, unmodified γ-PGA is more popular in
drug and parenteral vaccine delivery but it has not
been used on its own in nasal vaccine delivery.
Poly(l-lysine) is widely used alone or grafted with
other polymers to deliver DNA vaccines. The cat-
ionic functional group (-NH3 +) of lysine moiety effi-
ciently condenses anionic plasmid DNA into com-
pact nanoparticles [78] . Wu et al. reported the use of
poly(l-lysine) to deliver DNA vaccines to NALT. M
cells targeting recombinant protein sigma 1 (σ1) were
conjugated to polylysine and complexed with DNA vac-
cines [79] . Upon intranasal vaccination with this delivery
system in mice, higher levels of both humoral and cel-
lular immune responses were observed in comparison to
naked DNA vaccines.
Poly(l-arginine) is a safe, biodegradable cationic
poly(amino acids) and successfully applied in Phase
II clinical trials for hepatitis C virus-based peptide
vaccines [80] . Poly(l-arginine) along with dextran sul-
fate form polyelectrolyte capsules, which encapsulate
a large amount of antigens [81,82] . The cationic nature
of poly(l-arginine) nanoparticles enhances mucosal
membrane permeation of antigens [83] . De Koker et al.
showed that vaccination with poly(l-arginine)-based
capsule-encapsulating OVA antigens induced local
mucosal and cellular responses against adminis-
tered antigens [82] . While mucosal immunization of
poly(l-arginine) nanoparticles by the pulmonary route
has been reported, their use as a nasal vaccine delivery
system remains to be investigated [82] .
Other polymers
Other polymers such as polyanhydride and polyac-
rylates were also examined for intranasal delivery of
158 Ther. Deliv. (2017) 8(3)
vaccines. Polyanhydride nanoparticles were prepared
by the copolymerization of methyl vinyl ether and
maleic anhydride (PVM/MA). PVM/MA polymers
are considered safe and have been licensed for human
use [84,85] . Polyanhydrides showed a great potential in
drug or vaccine delivery attributed to their mucoadhe-
sive, controlled release and adjuvanting properties (as
they are Toll-like receptor (TLR)-2, TLR4 and TLR5
agonists) [84,85] . Gamazo and colleagues used outer
membrane vesicles (OMV) derived from Shigella flex-
neri as the antigen component and encapsulated it into
polyanhydride nanoparticles [86] . When tested in mice
intranasally, OMV-loaded polyanhydride nanopar-
ticles enhanced antibody titers, IFN-γ and IL-10 pro-
duction. Upon challenge in mice with S. flexneri 2a,
nanoparticle-vaccinated groups showed 100% survival
rate for up to 15 days indicating the enhanced protec-
tion capacity of OMV-encapsulated nanoparticles.
Polyacrylates were used as a vaccine delivery system
due to their bioadhesive property and mucosal-adju-
vanting effects [87–89] . Recently, Zaman et al. prepared
a polyacrylate-based delivery system by conjugating
polyacrylate dendrimers with peptide antigens [87] .
Upon intranasal immunization, polyacrylate-based
nanoparticles induced systemic antigen-specific IgG
responses; however, mucosal IgA antibodies were not
observed.
Lipid-based nanoparticles
Antigens can be delivered using varieties of nontoxic
and biodegradable lipids. Lipid-based nanoparticles
are popular vaccine delivery systems and some of them
have already found application in the commercialized
vaccines. Most of these particles showed mucoadhesive
properties. However, in contrast to chitosan, they were
unable to open tight epithelial cell junctions in NALT
for antigen transportation across the membrane [90] .
Liposomes
Liposomes were first reported in the mid-1960s. Cur-
rently, they are one of the most successful drug and
vaccine delivery systems. Several liposome-based prod-
ucts are already in the market and under clinical tri-
als [91] . Liposomes are spherical self-assembled lipid
bilayer vesicles with an aqueous core [92] . This unique
feature of liposomes enables encapsulation of both the
hydrophilic or lipophilic agents into their aqueous core
or lipid bilayer surface, respectively. Liposomes are usu-
ally prepared from natural or synthetic phospholipids
with or without the addition of cholesterols. Cholester-
ols stabilize the liposomes’ bilayer by enhancing their
rigidity. Classical neutral liposomes possess several
advantages when delivered parenterally; however, the
mucosal delivery of liposomes is often problematic due
future science group

to their low mucoadhesiveness or the reduced capac-
ity of liposomes to permeate the epithelial membrane.
These shortcomings have been overcome by using cat-
ionic liposomes or modulating the surface properties of
liposomes with cationic polymers.
Cationic liposomes are known as a safe and potent
mucosal delivery system [93] . Recently, Tada et al.
showed that the intranasal administration of OVA-
loaded cationic liposomes promotes antigen uptake
by NALT DCs [94] . Upon intranasal immunization in
mice, cationic liposomes induced an enhanced immune
response (IgA and IgG antibody titers) against OVA
compared with soluble antigen, anionic and neutral
liposomes. Slütter et al. reported contradictory find-
ings on the benefit of using cationic liposomes as a
nasal vaccine delivery system [95] . Efficiency of three
different formulations: OVA antigen, OVA+CpG and
OVA/CpG/liposomes was compared upon intranasal
immunization in mice. OVA/CpG-loaded liposomes
had the highest uptake by DCs in vitro, while they
showed poor uptake in vivo in mice in comparison
to OVA+CpG. Moreover, the serum IgG responses
in mice upon intranasal immunization for liposomes
loaded with OVA+CpG were lower than OVA+CpG.
Mucosal antibody responses were not reported. How-
ever, due to the use of very strong adjuvant (CpG)
and lack of proper control (OVA-loaded liposomes), it
could not be concluded that cationic liposomes lack
adjuvanting effects. In addition, except where men-
tioned above, OVA/CPG/liposomes’ as alternative
delivery system utilizing cationic liposomes showed
clear benefits of this formulation [96,97] .
The efficiency of a liposome-based nasal vaccine can
be improved by modifying its surface functionality.
Among various pathogen-recognizing receptors, APCs
express C-type lectin receptors that recognize the car-
bohydrates domain and efficiently promote antigen
presentation on MHC I and MHC II molecules [98] .
APCs can be targeted using carbohydrates on the
surface of liposomes [98,99] . Wang et al. prepared lipo-
somes surface-modified with carbohydrate domain-
galactose [99] . The OVA-loaded galactosylated lipo-
somes were efficiently endocytosed by macrophages
and produced a significant amount of systemic and
mucosal antibody titers upon intranasal immunization
in mice, compared with free antigens and unmodified
liposomes.
Liposomes can be additionally modified with poly-
mers such as chitosan, TMC or hyaluronic acids [92] .
The surface modification of liposomes with mucoad-
hesive polymers not only provides for the additional
protection of antigens against enzymatic degradation
but also allows them to retain antigens into the nasal
mucosa for prolonged periods of time, thus e­nhancing
future science group
Intranasal delivery of nanoparticle-based vaccines Review
interactions between particles and immune cells. In
addition, coated liposomes are usually more stable
under storage and physiological conditions. Lipo-
some–polymer hybrid nanoparticles were used to
deliver antigens or mixtures of antigens and immu-
nomodulatory agents [64,100] . Recently, liposomes-
bearing cationic moiety, 1,2-dioleoyl-3-trimethyl-
ammonium propane (DOTAP), were prepared for
encapsulation of F1-V antigen and MPLA [100] . These
cationic liposomes were electrostatically complexed
with anionic-thiolated hyaluronic acid and stabilized
with thiolated-PEG-forming DOTAP/HA core–PEG
shell nanostructures. These lipid–polymer hybrid
delivery systems (DOTAP/HA core–PEG shell nano-
structures), co-encapsulating adjuvant and antigens,
induced a high level of mucosal, systemic and cellu-
lar immune response upon intranasal immunization
in mice compared with soluble OVA and mixture of
monophosphoryl lipid A (MPLA) + OVA.
Emulsions
Emulsions are primarily isotropic mixtures of two
immiscible liquids (oil and water) stabilized by the
surfactants, and are capable of producing particle sizes
below 200 nm [101] . Nanoemulsion-encapsulating anti-
gens increase the stability of antigens against enzy-
matic degradations and control their release. The most
well-known emulsions used for vaccine delivery are
squalene-based o/w emulsions (MF 59 and AS03) that
have been applied in marketed vaccines [102] . How-
ever, their application is essentially limited to only the
p­arenteral route.
Makidon et al. studied a novel nanoemulsion formu-
lation based on soybean oil-in-water (o/w) for subunit
vaccine delivery [103] . The soybean-based nanoemulsions
were effectively internalized across the epithelial mem-
brane by the transcellular process and did not require
the opening of tight junctions or M cells’ participa-
tion for its enhanced permeation. This nanoemulsion
platform was used to develop a vaccine against hepati-
tis B [104] . HBsAg-loaded nanoemulsion was tested for
its efficacy in different animal models using intranasal
administration. Robust systemic, mucosal and cellu-
lar antigen-specific immune responses were observed
and the serum IgG antibody titers were comparable
to those induced by alum-adjuvanted HBsAg upon
i­ntramuscular a­dministration in mice.
Nanoemulsions provide a mucoadhesive effect by
interacting with mucin and these interactions depend
on the properties of oils and surfactants used in the
formulations; for example, the hydrophilic-lipophilic
balance value and charge in the surfactants. The
overall charge in the nanoemulsion can be altered by
varying nanoemulsion compositions. By varying the
www.future-science.com 159
Review Marasini, Skwarczynski & Toth
s­urfactants or altering the ratio of surfactant mixtures
used in nanoemulsions, the type of immune responses
(Th1, Th2 and Th17) were altered [105] . Wong et al.
observed that a highly cationic nanoemulsion had
more affinity toward mucin than uncharged and
anionic nanoemulsion [106] .
Immunostimulating complexes
Immunostimulating complexes (ISCOMs) are self-
assembled cage-like structures with icosahedral sym-
metry composed of antigens, QS, phospholipids and
cholesterol [107] . They are usually 40-nm-sized parti-
cles and incorporate hydrophobic antigens into their
structure. The efficiency of antigens’ incorporation
into ISCOMs is mainly related to the strength of the
hydrophobic interactions of antigens and the phospho-
lipids [107,108] . ISCOMs-based vaccines incorporating
recombinant HBsAg were shown to enhance muco-
sal, systemic and cellular immunity upon intranasal
immunization [109] . More importantly, ISCOMs gen-
erate both Th1 and Th2 immune responses, while
the most popular cholera toxin generates only Th2
responses against administered antigens [108] .
Classical ISCOMs also known as Quil A® derived
from Q. saponaria contain saponins, which are toxic and
therefore their clinical application was restricted [110].
Consequently, the saponins were substituted with
less toxic analogs known as QB-90 obtained from
Q. brasiliensis. QB-90 induced comparable or higher
antigen-specific mucosal and systemic antibody titers
than Quil A [111,112] . Cibulski et al. prepared ISCOMs
comprising QB-90, phospholipids, cholesterol and
OVA, and studied their efficiency to induce immune
responses in mice after intranasal administration [111] .
Mice immunized with ISCOMs (QB-90) showed sig-
nificantly higher antigen-specific serum and mucosal
immunity at nasal and distal mucosal sites such as the
vagina and intestines, compared with antigens alone
and classical Quil A-based ISCOMs.
Particles delivered via the nasal route should pos-
sess low mucociliary clearance for efficient uptake in
the NALT. Pandey et al. used the gamma scintigraphy
technique to show that ISCOMs were retained in the
nasal cavity for at least 2 h, while the control sodium
pertechnetate solution was rapidly cleared [113] . The
hydrophobic property of ISCOMs was found to be cru-
cial for ISCOMs’ deposition in the nasal tract. Thus,
the use of mucoadhesive ISCOMs seems an alternative
for the intranasal delivery system for vaccines.
Lipopeptides
Lipopeptides are amphiphilic molecules containing sin-
gle or multiple copies of peptides covalently conjugated
to one or more lipidic moieties. The bacterial-derived
160 Ther. Deliv. (2017) 8(3)
lipopeptides such as tripalmitoyl-S-glyceryl cysteine
(Pam3Cys) and its derivative (Pam2Cys) are commonly
used to induce potent immunity against peptide-based
antigens [114] . The adjuvant activity of these bacterial
lipopeptides is due to the recognition of lipid moiety
by the TLR2, a pathogen-associated molecular pattern
receptor present on the APCs [115] . A comparison of
efficacy between Pam2cys and Pam3Cys showed that
Pam2Cys containing peptide epitopes were more immu-
nogenic than Pam3Cys containing peptide epitopes,
which was most likely related to the poor aqueous solu-
bility of Pam3Cys [116] . Jackson and colleagues observed
that upon conjugation of Pam2Cys or Pam3Cys into
the middle of a long peptide sequence (carrying B cells
and T-cell epitopes; lipopeptide 1, Figure 3), the formed
lipopeptides were more water-soluble than when lipids
were attached to the end of the N-terminal moiety of
the peptide antigen (lipopeptide 2) [116] . Upon intrana-
sal immunization in mice, the branched lipopeptide 1
(Figure 3) containing Pam2Cys as a lipid moiety showed
significantly higher systemic antibody titers than a
linear Pam2Cys containing lipopeptide 2. Intranasal
administration of Pam2Cys with influenza virus antigen
induced secretion of IL-2, IL-6, IL-10, IFN-γ, MCP-1
and TNF-α and protected mice against the influenza A
virus challenge [117] .
Lipopeptides can self-assemble in water to form
both nano- and microsized particles [118] . The self-
assembling property of lipopeptides relies on the
hydrophilic-to-lipophilic balance or interactions
among peptides and lipids [118,119] . Toth and colleagues
introduced self-adjuvanting lipopeptides, also known
as lipid core peptides (LCPs), as a vaccine candidate
against group A streptococcus infection [120,121] . The
LCP-based vaccine candidates are chemically synthe-
sized in a pure and defined molecular form by standard
solid-phase peptide synthesis using either Boc or Fmoc
chemistry. LCPs contain lipoamino acids conjugated
to lysine moiety(s) via an amino acids linker (usually
two serines). The lysine(s) serve as branching moiety
to which multiple peptide epitopes are attached [120] .
Toth et al. incorporated a B-cell epitope (J14) derived
from a highly conserved region of M-protein from
group A streptococcus infection and a universal
T-helper cell epitope (P25) into the LCP system. The
LCP construct improved antigen-specific mucosal and
systemic immune responses upon intranasal immuni-
zation [26,122] . They also studied the structure activity
relationship of spatial arrangements of epitopes and
lipids [26] . The lipopeptide 3 (Figure 3) elicited high
antigen-specific mucosal titers. In contrast, when
the position of epitopes and lipids were rearranged
(lipopeptides 4 and 5), the IgA antibody titers were
s­ignificantly lower.
future science group

Recently, Ghaffar et al. showed that the combina-
tion of cationic liposomes and lipopeptides provided
a synergistic effect on immune responses upon intra-
nasal administration in the mice model [96] . Different
lipopeptides, varying in the number of lipids/peptides,
were anchored to liposomes. After three intranasal
immunizations, lipopeptides bearing two lipids in
the final construct showed the highest and most long-
lasting mucosal and systemic antibody titers compared
with free lipopeptides (containing the same number of
lipid moieties), peptides encapsulated into liposomes
or lipopeptides-bearing single lipid moiety encapsu-
lated into liposomes. The immunogenic efficiency of
lipopeptide-loaded liposomes upon intranasal admin-
istration was further enhanced by coating of liposomes
with chitosan-based polymer [64] . Recently, cationic
lipopeptides were incorporated either on the surface or
encapsulated into PLGA nanoparticles [123] . Nanopar-
ticles-bearing lipopeptide antigens inside the PLGA
nanoparticles showed significant improvement in
inducing antigen-specific mucosal and systemic anti-
bodies production compared with lipopeptide-coated
PLGA nanoparticles.
Does size of the nanoparticles influence
nasal immune responses?
The role of size of nanoparticles in APCs’ uptake
efficacy, their transportation from injection sites to
the lymph nodes, their stability in the systemic cir-
culations and their potency to induce antigen-specific
immune responses are extensively reviewed else-
where [18,19,124,125] . However, these reviews focused on
vaccines administered by the parenteral route. Unlike
parenteral vaccines, intranasal nanoparticle-based vac-
cines primarily are required to cross mucosal barriers
before being available to APCs for further antigen
processing. Alonso and colleagues prepared different
sized PLA–PEG particles (200 nm, 1.5, 5 and 10 μm)
encapsulating radio-labeled model protein, TT [126] .
The 200-nm-sized particles demonstrated stronger
mucosal permeation ability and higher bioavailability
than microparticles.
While there is no doubt that parenterally admin-
istered small nanoparticles (10–50 nm) induce
humoral immunity more efficiently than larger size
nanoparticles, several contrasting reports on optimum
nanoparticle size for enhanced immunogenicity have
been reported for intranasal vaccines [125] . Recently,
Toth and colleagues showed that the immunogenic-
ity of lipopeptide-based vaccines relies on the size of
nanoparticles [20] . Three different LCP vaccine candi-
dates incorporating B-cell epitopes (J14 and LP-88/30)
and universal T-helper cell epitope (P25) were pre-
pared. These self-adjuvanting LCP vaccine candidates
future science group
Intranasal delivery of nanoparticle-based vaccines Review
Peptide 1 Peptide 2
Lipids 1 or 2 Lipopeptide 1
Lipids 1 or 2 Peptide 1 Peptide 2
Lipopeptide 2
Peptide 3
Lipid 3
Peptide 4 Lipopeptide 3
Peptide 4 Peptide 3
Lipid 3 Lipopeptide 4
Peptide 3 Peptide 4
Lipid 3 Lipopeptide 5
Figure 3. Schematic representations of synthetic lipopeptides with
different spatial arrangement of epitopes. Lipopeptides 1 and 2
contained Peptide 1: T-cell epitope (GALNNRFQIKGVELKS) obtained from
influenza virus hemagglutinin; Peptide 2: B-cell epitope (EHWSYGLRPG),
a leutinizing hormone-releasing hormone; and Lipid 1: Pam2Cys or Lipid
2: Pam3Cys homologous to gram-negative bacterial outer membrane lipid.
Lipopeptides 3, 4 and 5 contained Peptide 3: universal T-helper cell epitope
(P25: KLIPNASLIENCTKAEL) derived from canine distemper virus; Peptide
4: B-cell epitope (J14: KQAEDKVKASREAKKQVEKALEQLEDKVK) derived
from M-protein of group A Streptococcus; Lipid 3: two copies of synthetic
lipoamino acids (2-R/S-amino hexadecanoic acid).Pam2Cys: Dipalmitoyl-S-
glyceryl cysteine; Pam3Cys: Tripalmitoyl-S-glyceryl cysteine.
showed a diverse range of particle sizes upon self-
assembling into phosphate-buffered saline (5, 10 and
50–100 nm). The smallest size nanoparticles (5 and 10
nm) were efficiently taken up by APCs leading to high
APCs maturations in vitro. Furthermore, intranasal
immunization with small-size nanoparticles (5 and 10
nm) induced the highest systemic IgG antibody pro-
ductions compared with larger size particles (50–100
nm). However, production of mucosal antibody titers
was not reported. In another study, the role of particle
sizes after intranasal administration of lipopeptide/
liposomes vaccines was examined [97] . Various sizes
(70, 140, 400 and 150–1000 nm) of lipopeptide-loaded
liposomes were prepared. The smaller sized liposomes
www.future-science.com 161
Review Marasini, Skwarczynski & Toth
induced the production of higher mucosal IgA and
systemic IgG antibody titers than larger liposomes;
however, the difference was not statistically significant.
Tada et al. formulated different sized OVA-loaded cat-
ionic liposomes (50, 100 and 1000 nm) and studied
their influence in inducing humoral responses [94] . No
size-based correlation on antigen-specific mucosal IgA
and systemic IgG responses was observed. In contrast,
other studies showed that larger nanoparticles induced
better humoral immunity than smaller sized counter-
parts. For instance, Gutierro et al. observed that 1000-
nm sized BSA-encapsulated PLGA nanoparticles were
more efficient in inducing systemic immunity com-
pared with 500- and 200-nm particles, upon intrana-
sal administration. Their effect on mucosal immunity
was not reported [127] . Similarly, Stano et al. compared
efficiency of different-sized OVA-conjugated pluronic-
stabilized polypropylene sulfide nanoparticles (30 vs
200 nm) upon intranasal administration in mice [128] .
They showed that large nanoparticles (200 nm) with a
slight anionic charge (-6.5 mV) induced significantly
stronger mucosal and systemic antibody titers than
smaller nanoparticles (30 nm) with cationic charge
(+8 mV).
Apparently, many physicochemical properties of
nanoparticles, apart from the size, influence the per-
meation of mucosal membrane (e.g., lipophilicity,
molecular weight of polymeric nanoparticles as well as
their stability and charges) [129] . To properly examine
any size-immunogenicity correlation, other properties
of nanoparticles must be maintained constant, which is
very difficult to achieve in most cases. Thus, the role of
particle size in mucosal and systemic immunity upon
intranasal vaccination is still waiting for careful and
thorough examination. Taken together, at the current
stage, most studies report that small-sized particles
were more efficient in mucosal barrier permeation and
induction of immunity against administered antigens.
Conclusion & future perspective
Owing to the large number of pathogens that infect
through the mucosal surface, a mucosal vaccine deliv-
ery route is expected to be a popular nature-mimicking
path for vaccine administration. While parenteral vac-
cines induce only systemic immunity, mucosal vac-
cines are able to induce both the mucosal and systemic
immunity. This unique feature of mucosal vaccines
allows mucosal IgA antibodies to opsonize or bind
the pathogens at the primary site of pathogen expo-
sure (mucosa), thus preventing further systemic inva-
sions. Mucosal vaccines are particularly important for
blocking the spread of diseases from nonsymptomatic
patients carrying the pathogen on their mucosal tissue.
Unlike parenteral vaccines, the major hurdle for nasal
162 Ther. Deliv. (2017) 8(3)
vaccine development is the requirement to cross the
mucosal membrane to be available for DCs to activate
the immune cascade. As discussed in this review, poly-
meric or lipid-based nanoparticles that mimic the size
of pathogens are an attractive choice for nasal vaccina-
tion owing to their ability to permeate transcellularly
or paracellularly across the tight epithelial junctions,
and their potential to target M cells in the NALT
region. While there is no clear correlation between
size of nanoparticles and their mucosal immunoge-
nicity, most studies agree that nanosized particles are
more efficient in inducing immune responses than
microsized particles. Several other factors such as the
efficiency of antigen loading, surface charge, muco-
adhesiveness, antigen release profile, nature and the
composition of biomaterials influence overall immune
responses.
Polyesters (e.g., PLGA and PLA) and polysaccha-
rides (e.g., chitosan and its analogs) and self-adju-
vanting lipopeptides have shown promising potential
as mucosal vaccine delivery systems. However, deliv-
ery of antigens in nanoparticle-based delivery systems
intended for nasal administration need to overcome
several practical hurdles such as the possibility of anti-
gen denaturation by the use of organic solvents during
nanoparticle production, low antigen entrapment into
nanoparticles and potential cellular toxicity. Thus,
there is a manifest need to develop a nanoplatform
for nasal vaccines using particle-engineering strategies
that decrease these risks, maintain long-term antigen
retention in the nasal mucosa and prolong antigen
availability to interact with immune cells. Some of the
strategies include functionalization of the particle sur-
face by conjugating with antigens or M cells target-
ing ligands, and modification of the physicochemical
properties of the particles (optimal particle composi-
tion, charge, shape and hydro-/lipophilicity), etc.
There are long lists of clinical trials for nasal vac-
cines, while only a few nasal vaccines such as Flu-
mist (USA), Fluenz (EU), Nasovac (India) have been
approved for human use. However, no current clini-
cal trial exists for a nanoparticle-based vaccine in the
database of the US National Institute of Health (clini-
caltrials.gov) [130] . In vivo studies discussed in this
review are mostly based on preclinical data and cannot
be generalized to humans. However, animal model-
based investigations provide some promising forecasts
for future human studies with nanoparticles. The use
of safe, biodegradable, biocompatible and biologically
stable nanoparticles creates high hopes of translating
nasal nanoparticle vaccines from laboratory to clini-
cal use. The vaccine candidates should be well-defined
chemically and produced in its purest form to avoid any
toxicity issues. In the near future, novel conjugation
future science group
 Intranasal delivery of nanoparticle-based vaccines Review

strategies that combine synthetic peptide-based epi-
topes (single or multiple) with M-cell-targeting moi-
eties, TLR agonists lipids, carbohydrates, dendrimers
and polymers into single nanoparticles shall provide a
rational approach to successfully deliver nasal vaccines
and tailor the required immune responses.
Financial & competing interest disclosure
This work was supported by the National Health and Medical
Research Council (NHMRC), Australia. The author, N Marasini,
is a recipient of an international postgraduate research schol-
arship (IPRS) and Australian postgraduate award (APA) for his
PhD study. The contents are solely the responsibility of authors
and do not necessarily represent the official views of NHMRC.
The authors have no other relevant affiliations or financial in-
volvement with any organization or entity with a financial inter-
est in or financial conflict with the subject matter or materials
discussed in the manuscript apart from those disclosed.
No funded writing assistance was utilized in the production
of this manuscript.

Executive summary
Background
• Most pathogens gain access to the human body through mucosal sites and initiate systemic infections.
• Parenterally administered vaccines induce strong systemic immunity and almost no mucosal immunity.
• Nasal vaccination seems an appealing strategy for the induction of antigen-specific systemic and mucosal
immunity.
• A safer mucosal adjuvanting strategy or mucosal platform technology for intranasal immunization is much
warranted.
Nanoparticles as nasal adjuvanting system
• Particulate vaccines formulated into nanosize but not microsize showed more efficient APC internalizations,
cross-presentation and B- and T-cell responses.
• Adjuvanting effects of the nanoparticles are related to their improved ability to cross mucosal barriers,
enhanced uptake by APCs, prolonged availability to interact with APCs and provision of additional danger
signals due to their size mimicry of pathogens.
• Nanoparticles are functionalized by conjugating with antigens or M-cell-targeting ligands, and modification
in physicochemical properties of the particles (optimal particle composition, charge, shape and hydro/
lipophilicity, etc.) to enhance immunogenicity of administered antigens.
• The surface of nanoparticles can be modified by using mucoadhesive polymers (such as chitosan and their
derivatives) or imparting a cationic charge on the particle’s surface to improve mucus-binding efficacy and
nasal residence time.
Conclusion & future perspective
• The use of safe, biodegradable, biocompatible and biologically stable nanoparticles as nasal delivery systems
creates a high hope of translating nasal nanoparticle vaccines from laboratory to clinical use.

6 Mcentee C, Lavelle EC, O’hagan DT. Chapter 63 – Antigen

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