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Nanoparticulate mediated transcutaneous immunization: Myth or reality

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DOI: 10.1016/j.nano.2015.12.372

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 Nanomedicine: Nanotechnology, Biology, and Medicine
12 (2016) 1063 – 1081

Original Article nanomedjournal.com

Nanoparticulate mediated transcutaneous immunization: Myth or reality

Monika Kaurav, MPharm a , Sunita Minz, MPharm a , Kantrol Sahu, MPharm a , Manoj Kumar, PhD a ,
Jitender Madan, PhD b , Ravi Shankar Pandey, PhD a,⁎
a
SLT Institute of Pharmaceutical Sciences, Guru Ghasidas Vishwavidyalaya, Bilaspur, India
b
Chandigarh College of Pharmacy, Mohali, Panjab, India
Received 2 October 2015; accepted 17 December 2015

Abstract

Transcutaneous immunization (TCI) is a promising route of vaccine delivery through skin due to many well documented advantages. The
main obstacle in TCI is the skin's top dead layer i.e. stratum corneum which is difficult to penetrate. Efficiently delivery of antigen to the
immune competent cells of epidermis or dermis in TCI might elicit an effective immune response. In this review, skin immunology with a
particular focus on potential of immunological active receptors in influencing adaptive immune responses is highlighted. The challenges with
TCI and methods to improve it using different adjuvants, chemical and physical approaches, delivery systems, and combination of above
methods to further improve immune response following skin application of antigen are elaborately discussed. Nanoparticulate vaccine
delivery systems with reference to their applications in TCI are classified according to their chronological development. Conclusively,
clinical translations of above methods are also briefly reviewed.

From the Clinical Editor: Transcutaneous immunization has been investigated by many as a promising route of vaccination. In this
comprehensive review article, the authors described and discussed the existing knowledge and difficulties in this approach. Furthermore,
ways of improving transcutaneous delivery were also reviewed.
© 2016 Elsevier Inc. All rights reserved.

Key words: Transcutaneous immunization; Adjuvants; Nanoparticulate delivery systems

Vaccination has been one of the extensively booming medical
interventions in diminution of infectious diseases since the
nineteenth century. 1 According to definition, a good vaccine
should be safe, causes minimal pain if invasive and most
importantly elicits long lasting strong and protective immune
response. 2 Activation of immune system is the main concern to
be faced in vaccine development. Due to the limited accessibility
of efficiently active antigen presenting cells (APCs) at the
location of vaccination of traditional vaccine method, represen-
tation of vaccine originated antigen to the immune system is
reduced which makes it insufficient process. 3
Conflicts of interests: The authors declare no conflict of interest.
⁎Corresponding author at: SLT Institute of Pharmaceutical Sciences,
Guru Ghasidas Vishwavidyalaya, Bilaspur, India.
E-mail addresses: monika11kaurav@gmail.com (M. Kaurav),
sunitaminz.3481@rediffmail.com (S. Minz), kantrol.sahu23@gmail.com
(K. Sahu), mrmanojkumar1@yahoo.co.in (M. Kumar),
jitenderpharmacy@gmail.com (J. Madan),
ravishankarpandey2@rediffmail.com, rspandey1@gmail.com (R.S. Pandey).
http://dx.doi.org/10.1016/j.nano.2015.12.372
1549-9634/© 2016 Elsevier Inc. All rights reserved.
Traditionally available vaccine delivery methods are oral
(OR), intranasal (IN), intradermal (ID), intramuscular (IM) and
subcutaneous (SC). 4 Out of these IM, SC and ID immunizations
are classified as needle-based techniques. Although needle-based
techniques are effective in achieving the desired immune
response, however their use has numerous limitations such as
injection requires sterilized needles and skilled people. More-
over, injection can be irritating and painful, particularly in
children. 5 The muscular site, a conventional site of immuniza-
tion, is not regarded as to be a proficient site for antigen
presentation owing to lack of suitable density of APCs dendritic
cells (DCs) or macrophages. 6 However, antigens alone usually
are inadequately immunogenic and hence necessitate to be
prepared with adjuvants to amplify DC establishment and
infiltration into the intramuscular immunization sites, 6 -9 finally
from all above, adding to the belief that the muscles and SC
routes are not an ideal site meant for immunization. The
drawbacks of injectable vaccines have heighten the research on
oral, 10 nasal, 11 pulmonary 12 and transcutaneous 13 deliveries of
vaccines. Despite the efficacy of traditional (attenuated or killed)
vaccines, there is concern over their safety, 14 represented in
Figure 1, along with limitation of injectable vaccines. 15

Please cite this article as: Kaurav M, et al, Nanoparticulate mediated transcutaneous immunization: Myth or reality. Nanomedicine: NBM 2016;12:1063-1081,
http://dx.doi.org/10.1016/j.nano.2015.12.372
1064 M. Kaurav et al / Nanomedicine: Nanotechnology, Biology, and Medicine 12 (2016) 1063–1081
Figure 1. Disadvantages of traditional vaccines and limitations of injectable vaccines.
Skin is abundant in trained APCs such as DCs in the dermis
and Langerhans cells (LCs) in the epidermis, these cells can
professionally activate both in vitro and in vivo primary immune
responses. 16,17 Thus TCI is one of the most promising
immunization methods. Vaccine administration via the skin
without any pain by means of a larger surface area is referred to
as transcutaneous immunization (TCI). The transcutaneous route
is principally striking because the skin is vastly accessible and
has distinctive immunological characteristics, 18 while for a long
time, it has been acknowledged that an effectual immune
response can be induced through the skin and various diverse
approaches have been attempted. One of victorious examples of
TCI is a scarifying skin in the case of vaccinia virus vaccination
in humans. 19 The existence of skilled APCs in the epidermis and
the dermis mediates the immune response following cutaneous
immunization. 16 An additional crucial rationale for considering
the transcutaneous route is the prospective for protected immune
stimulation, as it circumvents the straight contact between
persuasive adjuvants with the general circulation. 20 The
advantages associated with TCI listed in Table 1. 21,22
Immunological mechanism system of skin and initiation of
immune response
Foremost components of skin immune system are keratino-
cytes in the viable epidermis while melanocytes, Merkel cells
and Langerhans cells are less abundant and present in dermis.
Keratinocytes constitute about 90% of total epidermal cell
population playing important role in innate immunity of the skin.
In case of skin barrier interruption, keratinocytes produce many
types of chemokines, cytokines and antimicrobial proteins and
peptides [eg. tumor necrosis factor-α (TNF-α), interleukin-1α
(IL-1α), interleukin-1β (IL-1β) and granulocyte macrophage-
colony stimulating factor (GM-CSF)]. 23 These chemokines are
key mediators in the generation of innate immunity upon
interaction with DCs. 24 Besides keratinocytes, other cells
involved in cytokine secretion and DC maturation are natural
killer cells, mast cells, macrophages, and neutrophils. 25 The skin
in addition has significant immunological roles owing to the
presence of the distinctive skin-associated lymphoid tissues
(SALT), 26 which is constituted by skilled APCs, such as LCs
and dDCs (dermal dendritic cells) along with other skin cell
subsets of T-lymphocytes and lymph nodes, although of the total
skin cells, LCs detail for only 1% of those present near the
stratum corneum, outermost layer of the skin, and indicate a
system of immune cells that bring about 25% of the total skin
area and can transport antigen from the skin into the lymph node
to elicit immune responses. 27 The immune cells and immune
responses of skin immune system are represented in Figure 2.
DCs as a link between innate and adaptive immunity
As discussed before, skin DCs are an important linkage
between innate and adaptive immunity. They tutor and excite B
and T lymphocytes with the contribution of central role in both,
cell mediated as well as humoral immunity. Adaptive immunity
starts when DC cells matured and migrated to lymph node where
it interacts with T cell, resulted in the proliferation of B and T
lymphocytes in the spleen and lymph nodes that are secondary
lymphoid organs. Both these cells are developed from common
lymphocyte progenitor in bone marrow. DCs are another major
imperative APC in the skin and take part in a vital function in
both the innate and adaptive immune responses. Immature DCs
are activated by agents such as microbes, cells of immune
systems. Pathogen recognition receptors (PRRs) are agents that
initiated these responses by binding to them. Even though PRRs
are expressed on several cell types, DCs have an important role
in controlling immune response, so focus of DC activation
concentrated on PRRs. 17 The mechanism of action of PRRs
upon binding of an antigen with adjuvant is shown in Figure 3.
PRRs are further divided into three major classes, RIG-I-like
receptors (RLRs), NOD-like receptors (NLRs) and Toll-like
receptors (TLRs). 28
TLRs are one of the mainly imperative PRRs and provide
defense to host against a range of pathogens. On the basis of
selective recognition to diverse bacterial, viral pathogen or
endogenous signals, ten TLR members have been identified in
human. 29 These TLRs on the basis of their ligand recognition
and localization either on cell surface or intracellular, can be
further divided into subfamilies. In this subfamily of TLR 3, 7, 8
and 9 recognize nucleic acids, whereas the subfamilies of TLRs
1, 2, 4 and 6 recognize lipids. 30 TLRs 1, 2, 4, 5, 6, 10 and 11 that
detect bacterial products other than nucleic acid are present on
the cell surface, whereas TLRs 3, 7, 8 and 9 that detect nucleic
acids are located intracellularly, either on late endosomes or
lysosomes. Such limited localization possibly will afford the
mechanism by which DCs evade unprompted activation by self
nucleic acids. 31
 M. Kaurav et al / Nanomedicine: Nanotechnology, Biology, and Medicine 12 (2016) 1063–1081 1065

Table 1
Advantages of transcutaneous vaccines. 21,22
Non-invasive Does not require use of needle
Avoid rise of infection Avoid problem of blood transmissible infection due to needle re-use associated with injectable vaccine
Stable No first pass metabolism thus provide protection to antigen from enzymatic digestion
Better patient compliance Self administration possible, no need of trained personnel
Induction of immune response Stimulation of innate immune system that trigged appropriate adaptive immune response and induction of immunological
memory, activates LCs and DCs which induce Th1, Th2, cytotoxic T lymphocytes and different subclasses of IgG, IgA etc
No strict purity concern Given via skin route not in systemic so sterilization of formulation is not required
Easy and fast to produce Mass production of skin vaccine may be less expensive than that of injectable vaccine that requires high standard of purity
with sterility
Low cost of production Low cost due to no strict purity and sterility, increase usage and demand in developing countries
Protective barrier function Avoid risk of invasion by microorganisms, prevention of pathogen entry Into systemic circulation by local immune response
and prevention of infection spread
Effective in children, in aged and Easy withdrawal possible in case of side/adverse effect as compared to injectable vaccine and more attractive due to self
immunocompromised persons applicable

Most studies which are related to skin focused on TLR
expression on LCs and dDCs, which is divergent and
furthermore diverges from further subtypes of DCs at mucosal
outside or in the blood circulation. The TLRs and their natural
ligand location on skin immune cells (human and mouse) are
listed in Table 2. 29,31-34 The TLR distribution on DCs and the
difference in the epithelia micromilieu may persuade the immune
alteration function of definite adjuvants and in that way the
choice of adjuvants for TCI. 35
Transcutaneous immunization (TCI)
size which limit their transport across stratum corneum. 40 Apart
from this, a number of adjuvants are too toxic in nature for
clinical use. Adjuvants established with TCI are bacterial
exotoxins which are responsible for different cytokines expres-
sion from re-stimulated lymphocytes and TLR ligands. 41,42 To
elicit strong immune response with reduced nonspecific adverse
inflammatory response, there must be a need of optimal adjuvant
which targets LCs in skin eliciting systemic antibodies including
IgG and secretory IgA responses in both mice and humans.
Table 3 briefly discusses promising adjuvants established safe in
preclinical trials of TCI. 43 -53

As revealed before, TCI vaccine delivery through which
antigen topically applied on epidermis, now becomes a rising
antigen delivery methodology due to the fact that skin is
exceedingly accessible and has inimitable immunological
distinctiveness. 18 However, topical antigen application perme-
ation through skin into epidermis is restricted by the impervious
obstruction role of the stratum corneum, the outermost layer of
epidermis. 36 To induce efficient TCI, main challenges are
co-administration of adjuvants with antigen(s) and permeation
through skin barriers to induce strong immune response via
uptake of antigen by stimulated DCs, their maturation and
relocation in an appropriate manner.
Adjuvants used in TCI
In immunology “adjuvants” are agents that stimulate the
immune system and increase the response to the vaccine,
however with or without any antigenic effect in itself. In
combination with antigen adjuvant can enhance, accelerate or
prolong antigen specific immune response. 37 Adjuvants are one
of most important components in TCI, because response is
adjuvant dependent. Co-application of adjuvant with antigen is
required to elicit thriving immune responses by TCI. 38 The
variety of prospective adjuvants for vaccine is wide which
include microbial bacterial and toxin products, oligodeoxynu-
cleotides, cytokines, liposomes and mineral salts. 39 Aluminium
hydroxide, calcium phosphate, stearyl tyrosine and MF 59
emulsions are undesirable for TCI; due to their relatively large
Skin permeation enhancement approaches for TCI
Protein, peptides and vaccines are not suitable candidates for
oral delivery because they are prone to high degree of first pass
metabolism in the liver. Skin as a largest and most accessible
organ of the body provides a suitable site for administration of
above candidates. Transcutaneous delivery routes offer promis-
ing site by target delivery to immune cells such as LCs and
dendritic cells in the skin, can improve immune response and has
fascinating rising significance. Recently, advancements in
pharmaceutical biotechnologies and emerging improved tech-
niques and instruments can overcome problems related with the
stratum corneum barrier. In the direction of overcoming skin
barrier to non-invasive delivery, various sophisticated delivery
systems, passive as well as active approaches to optimize
transcutaneous delivery of protein and peptides have been
developed. All the vaccine candidates used for TCI administra-
tion do not have ideal physiochemical properties, so there is a
need to add some chemicals or vehicle or devices or formulations
to help them penetrate or cross the stratum corneum to get the
desired therapeutic response. Various approaches are discussed
below (Figure 4):
Physical approaches
As previously reviewed, penetration of substances in the skin
is restricted by the SC which acts as a physical barrier, so the
topical application of the vaccine formulation alone restricts its
1066 M. Kaurav et al / Nanomedicine: Nanotechnology, Biology, and Medicine 12 (2016) 1063–1081

Figure 2. Skin immune system. The skin is enriched with immunocompetent cells such as LCs, keratinocytes and several dDCs. Keratinocytes induce innate
immunity. LCs and DCs capture external Ag. Migrate into lymph node where they present Ag to T cells and activate Ag specific T cells and B Cells. Activated B
cells and T cells migrate to other parts and induce Ag-specific immune responses.

passage across the skin and hence efficient immune response is
not achieved. Various physical approaches such as electropora-
tion, iontophoresis, abrasions, tape stripping, sonophoresis,
micro needles etc have been investigated to increase the
penetration of substances across the skin by disrupting SC.
Another approach to bypass the barrier function of SC is
application of microneedles. 54 A microneedle array contains
many micrometer-sized needles that can create a transport
pathway large enough for proteins and NPs, but small enough to
avoid harm. 55,56 Tape-stripping method has been shown to
enhance cytotoxic T cells and cytokine mediated immune
responses upon subsequent application of various antigens and
adjuvants to the skin in mice. 57 Similarly electrode preparation
pads, emery paper, rubbing gauze or porous rock on the skin take
out cells by their scratchy effects and have been found to enhance
immune responses in humans. 58 Mild hyperthermia with
abrasion enhances transport of antigen into the skin, presents it
to LCs, enhances migration of activated LCs to lymph nodes, and
triggers cascade of immune system.
Chemical enhancer approaches
Chemical moiety which reduces the barrier function of the
stratum corneum is known as penetration enhancers. The
enhancer can either disrupt lipid organization and enhance
drug diffusion coefficient or interact with keratin in corneocytes,
thereby opening the dense protein structure. 59 In addition, via
increases of the partitioning of drugs, chemical enhancers result
in an increased diffusion rate. 60,61 More than 300 penetration
enhancers are known and categorized into different categories
based on their mechanism of penetration enhancement. Pre
hydration using alcohol swab can enhance the immune response
elicited by TCI. Scharton-Kersten reported that antibody titers
against CT (cholera toxin) were improved when the area of
immunization was swabbed with isopropanol prior to application
of the antigen. 62 Karande 63 formulated several formulations of
commonly used chemicals, screened their potency (delivery of
antigen) in vitro and subsequently tested for their adjuvancy
(activation of immune response) followed by investigation in
vivo for their aptitude to produce antibodies in opposition to
ovalbumin which is applied topically and shows to be
extensively more effectual in producing anti-IgG titers of
ovalbumin. Many delivery systems such as liposomes, dendri-
mers, and microemulsions because of their supramolecular
structure have also been used as chemical enhancers that increase
skin permeability and solubilization of medicaments in the
formulation and their partitioning into the skin. 64,65 These results
have shown that formulations prepared with chemical enhancers
can be advantageously used to deliver antigens through the
transcutaneous route.
Novel approaches for TCI using particulate antigen delivery
systems
Formulation development of antigens in particulate carrier
systems is a fascinating approach to advance vaccine delivery,
also through the transcutaneous method. 66,67 It is known that
disruption of skin barrier increases the transcutaneous perme-
ation of antigen and makes it easy to be taken up by antigen
presenting cells. So that non-invasive techniques that are capable
of disrupting the epidermal skin barriers have been widely
investigated to activate the immune system and by cytokines
persuade the secretion of pro-inflammatory keratinocytes and
promote proceeding of immune stimulatory events. Recently a
lot of attention has been focused on micro/nanometric delivery
systems such as patches, nanoparticles (NPs), liposomes,
 M. Kaurav et al / Nanomedicine: Nanotechnology, Biology, and Medicine 12 (2016) 1063–1081
Figure 3. Mechanism of action of pattern recognition receptors (PRR) upon binding of antigen with adjuvants used in TCI.
transferosomes, solution, emulsions, suspensions, niosomes,
cubosomes etc. Main priority is to formulate formulation to
target antigen to APCs and to protect antigen from degradation
for prolonged and long lasting effect.
In present review, advanced and novel topical delivery
systems have been categorized into three generations of
chronological development depending upon their utilization for
topical vaccination purpose. First generation topical delivery
systems have seen a sturdy increase in clinical use for delivery of
vaccines that are permissible to the skin via passive diffusion.
Work in this area progressed before 90s such as liposomes,
polymerosomes, niosomes NPs, patches etc. Second generation
delivery systems include systems with some more advancement
which emerged in 1990 to 2005 such as transferosomes,
ethosomes, cubosomes, vesosomes, immunostimulatory com-
plexes (ISCOMs), dendrimers etc. Sometimes they are used
along with chemical enhancers to enhance permeability and
drive transcutaneous transport and have also led to clinical
products. Third generation delivery systems emerge from 2005
to present and include inorganic NPs such as gold, magnetic,
quantum dots, carbon nanotubes etc. Schematic diagram of
several transcutaneous antigen delivery technologies for TCI is
shown in Figure 5 and briefly listed about different generation
delivery systems consisting about mechanism of action,
properties, limitation along with their utilization for TCI in
Table 4.
First generation delivery systems (before 90s)
Liposomes
Liposomes are spherical artificial phospholipid vesicles. They
assemble impetuously into bilayered arrangements including an
1067
inner aqueous core. 68 Liposomes' main component, phospho-
lipids are biocompatible and eco-friendly and these qualities
make liposomes a protected system, competent to be used in the
pharmaceutical ground. 69 They can result in prolonged primary
activation of T cells in vivo via averting the degradation of
antigens. 67 To deliver the molecule at the site of action, the lipid
bilayers fuse with other bilayer such as cell membrane delivering
the liposomal contents. 70 Tyagi et al, 71 reported the immu-
ne-enhancing effects of elastic liposomes consisting merozoite
surface protein-1 of Plasmodium falciparum through transcuta-
neous delivery. Mishra et al, 72 have studied that hepatitis B
surface antigen (HBsAg)-loaded elastic liposomes were an
effective mode for enhanced immunity against the antigen via
transcutaneous route.
Nanoparticles (NPs)
NPs are striking means for transcutaneous antigen delivery.
The underlying mechanism is that by disrupting SC as a result,
nano-bio interaction with skin lipid, the antigen encapsulated in
NPs delivered to the SC into the skin via induction of transient
and reversible opening of the stratum corneum. Just because they
seem alike in structure and size to the natural pathogen, which
are dynamically taken up by the APCs, can able to improve
antigen uptake. 73 An additional lead is the prospect to
encapsulate antigen and adjuvant together in the same particle,
which is put forward to improve the immunogenicity. 74
Additionally, it was proved that NP vaccine can penetrate hair
follicles where high density of APCs are present and target the
carried antigen toward APCs ultimately stimulate immune
responses. 75,76 Several numbers of polymers have been reported
for preparing NPs and induce well-built TCI. Schematic diagram
shows generation of adaptive immune response following topical
1068 M. Kaurav et al / Nanomedicine: Nanotechnology, Biology, and Medicine 12 (2016) 1063–1081

Table 2 interacting with a negative charge in the skin to improve antigen

Toll like receptors with their natural ligands and localities. 29,31-34 diffusion into the deeper layer of skin. 82 Chitosan and its
TLR Ligands Ligand locality derivative have several advantageous properties such as
TLR1 + TLR2 Tri-acyl lipopeptides Bacteria
biodegradability, biocompatibility, low toxicity and mild
TLR2 + TLR6 Di-acyl lipopeptides Mycoplasm preparation method made chitosan ideal for development of
TLR2 Glycolipids Bacteria drug delivery system. 83,84 N-trimethyl chitosan (TMC) deriva-
Lipoteichoic acid Bacteria tive of chitosan is water soluble and bears a permanent positive
HSP60, 70 and 90, grp96 Host cells a charge over a wide pH range which prominently increases the
TLR3 Double-stranded virus antigen transdermal penetration in vitro and in vivo. 85 TMC
RNA (dsRNA)
could appreciably modify the secondary structure arrangement of
TLR4 LPS Gram negative
bacteria keratin in the stratum corneum, increase the water content in the
HSPs Bacteria and host cells stratum corneum, enhance cell membrane fluidity, has intrinsic
Fibrinogen, heparin Host cells a adjuvant properties and trigger DC maturation. 86 Several reports
sulfate fragment demonstrated that chitosan NPs act as adjuvant, can also function
Hyaluronic acid as a depot 87 and are efficiently taken up by DCs than plain
fragment, β-defensin antigen and lead to the higher immune response than
TLR5 Flagellin Bacteria
TLR7/8 Single stranded RNA (ssRNA) Virus
intramuscular injection. 18 Li et al, 88 prepared chitosan NPs
TLR9 Unmethylated CpG DNA Bacteria loaded with ovalbumin or gp 100 protein which has shown
TLR10 Unidentified Unidentified significantly higher anti-OVA IgG titers and stimulated tumor
TLR11 (mice only) Profilin Toxoplasma gondii immune response for respective NPs after TCI.
TLR12 (mice only) Unidentified Unidentified
TLR13 (mice only) Unidentified Unidentified Niosomes
a
Danger associated signals molecules (DAMPs).
Niosomes are another type of delivery system that has been
reported in application of noninvasive TCI, having similar
physical properties and preparation method that of
application of antigen containing NPs with or without adjuvant liposomes. 89,90 For target delivery to the LCs niosomes made
(Figure 6). up of Span 60, Span 85, cholesterol and stearylamine containing
BSA were coated with O-palmitoyl mannan which is a modified
Polymeric PLA/PLGA NPs
polysaccharide. This niosomal formulation has shown notable
Commonly used PLGA/PLA NPs are extensively studied by higher level of serum IgG titers in compared to alum absorbed
several investigators for transcutaneous antigen delivery. When BSA and plain uncoated niosomes upon TCI administration, but
applied to the skin nano- and micron sized particulate carriers are less than those achieved with an equivalent dose of BSA-alum
mount up in the follicle ends, sebaceous glands or skin layers. after IM injection. 91 Maheshwari et al, 92 reported that cholera
The recent work of Hansen and Lahr 77 has shown that massaging toxin B when co-administered with the niosomes loaded with
of aqueous NP suspension as well as a hydrogel formulation of HBsAg topically is a potential adjuvant for cutaneous immuni-
NPs, into pig ear skin in vitro permeates in deep layers and into zation and it also explored niosomes vaccine delivery systems
hair follicles more compared to simple aqueous solution. They can be effectual as topical delivery of vaccines. Van den Bergh et
prepared NPs with polylactic-co-glycolic acid (PLGA) which is al, 93 prepared many formulations of surfactant-based elastic
biocompatible and biodegradable polymer along with a stabilizer vesicles, made up of a sucrose-laurate ester which is a
polyvinyl alcohol. Furthermore such particles may efficiently bilayer-forming surfactant and octaoxyethylene-laurate ester
able to release diverse loaded compounds that enter in the also a surfactant and sodium bis-tri-decyl sulfo-succinate
skin. 78,79 Mattheolabakis et al, 80 examined the potential of OVA which is a charge inducer. Ding et al, 94 prepared pH sensitive
loaded PLA NPs as a vector for antigen delivery via diphtheria toxoid loaded elastic vesicles. Results have shown
transcutaneous route to induce efficient cytokine production. In that vesicles that had antigen preserving potential and release
vitro culture of splenocytes with OVA re-stimulate thus elicited a antigen at desire pH make them excellent carrier for future
little higher levels of IFN-γ and IL-2 (P b 0.001) in mice, studies.
compared to those immunized with OVA in solution. Vogt et
al, 76 further investigated that smaller size NPs (40 nm NPs, but
Patches
not 750 or 1500 nm NPs) may be efficiently used for
transcutaneous delivery of vaccine compounds via the hair
Needle-free delivery has become a global priority because of
follicle into cutaneous APCs.
the hazard of needle-borne diseases associated with re-use and
Chitosan NPs inappropriate disposal of needle. Patch based vaccination has
been demonstrated for both promoting the penetration of
The properties of chitosan and its derivatives are promising in antigenic proteins across the stratum corneum and inducing
the application of TCI. Recently, they are reported as potential Ag-specific humoral immune responses, which neutralizes viral
transdermal penetration enhancer and their NPs were reported as infection and bacterial toxins in a safe manner. The underlying
an immune enhancer in TCI. 81 Chitosan appeared to be mechanism is hypothetical in that the antigenic proteins present
Table 3
Immune adjuvants used in transcutaneous immunization.
TCI adjuvants/resources Molecular mechanism
CT, CT-B A non hazardous B subunit with
strong binding affinity on GM 1
ganglioside receptors, which
presents on skin and mucosal
epithelial cells, lymphocytes and DCs
LT, LT-B Similar to CT induce monocyte-derived
DCs, fabrication of the CCR7, which
is expressed on mature DC that habitat
to the lymph nodes and production of IL-12
LPS and its natural and LPS induces an IL-10-driven
synthetic derivatives inhibition of CD4 T-cell
(MPLA)/the outer expansion and has role in IL-10
membrane production through monocytes
of Gram-negative subsequent to attachment of
bacteria PD-1 by PD-L1. LPS is the most
Detoxified derivative potent endotoxin that induces
of LPS from production of tumor TNF-α by macrophages
Salmonella minnesota LPS and MPLA both are TLR-4 agonist
CpG ODN/bacterial CpG ODN binds with
DNA and synthetic TLR9 located in endocytic
oligo-deoxy nucleotides vesicles. Unmethylated CpG
motifs directly trigger an
immuno-stimulatory
surge that concludes in the
maturation, differentiation,
and propagation of multiple immune
cells, including B and T
lymphocytes, monocytes, DCs,
NK cells and macrophages
Imiquimod Triggers skin immune cells via
TLR 7 and TLR 8 receptors,
also categorized as immune
modifiers, anti tumor
and antiviral effects
Cytokines: (IL-2, IL-12, Augmentation of
GM-CSF, Flt3) antibody responses
CT: Vibrio cholerae cholera toxin; CT-B: B subunit of cholera toxin; LT: heat-labile enterotoxin of Escherichia coli; LT-B: heat-labile enterotoxin of E. coli B subunit; LPS: lipopolysaccharide,
MPLA: monophosphoryl lipid A; IL: interleukin;
GM-CSF: granulocyte macrophage colony-stimulating factor; Flt3: fms-related tyrosine kinase 3 ligand.
Functions in TCI
Raise LCs population in epidermis;
induce antibody responses and
Th 1 and Th 2 mediated cytokines
production; CT-B potentiate
Th1 related immune responses;
produce ILs 2,4,5 and IFN-ү
Analogous structure and activities
to CT; improved uptake and
enhanced elicitation of response
to co-administered antigens;
produce ILs 2, 4, 5 and IFN-γ
Induces migration of LCs; raises
T cell activation; improves the
degree and extent of immune
responses; stimulates the host
cells to produce ILs and TNF.
By improving the speed, degree,
time period and affinity of the
defensive response, increases
the vaccine's immunogenicity;
promote production of
Th1-driven response; generate
IFN-ү and proinflammatory
cytokines and the
maturation/activation of
professional APCs
Convinces route of LCs; adapt
the immune response with
potent antiviral and anti-tumor
possessions; a strong inducer
of IFN-α, TNF-α
and ILs GM-CSF
– Flt3 along with HBV
Benefits Safety/immunogenicity Reference
43
Augmentation of level of Typical mucosal adjuvants, competent
IgA in serum and mucosa in several animal models, but toxic in
humans
M. Kaurav et al / Nanomedicine: Nanotechnology, Biology, and Medicine 12 (2016) 1063–1081
44,45
LT stimulates LCs cells, Rashes at local areas while applied as patch
rooting resettlement
from skin to draining LNs.
46-48
Along with polysaccharide-subunit Promising effects on neuro-inflammation
vaccine, developed Th1 and auto immune disorders being predicted.
responses to the carrier Strong prompter of pro-inflammatory cytokines
protein, with higher MPLA induce mild local reaction thus used as
antibody response safe cutaneous adjuvant
in animal but in humans
Phase 1 trials performed – 49,50
in humans (in involvement
with Alum) have shown
improved anti-HBsAg
antibody responses
Now oligonucleotides
classified into 3 classes
in consideration to their
distinct aptitude to trigger
either: human B, NK or
DCs in vitro.
Can stimulate DCs and B cells to – 51,52
induce Th 1 cell immunity, thus
boost up antibody production drained
out strong immune responses via microneedle
delivery along with influenza subunit vaccine
more than vaccine alone in skin mediated
influenza vaccine
Exploitation of cytokines in cancer 49,53
antigen induce the gathering of patients as Immuno adjuvant property
immature DCs in marginal blood, with some limitations such as some
without enhancement severe toxicity and
of antibody responses. short biological half-life.
1069
1070 M. Kaurav et al / Nanomedicine: Nanotechnology, Biology, and Medicine 12 (2016) 1063–1081
Figure 4. Schematic diagram of different approaches used to enhance skin permeation for TCI including physical, chemical, delivery systems and combination methods.
in concentrated amount on the patch surface might generate a
high antigenic protein concentration gradient, which is signifi-
cant for producing the driving force to hasten antigen
penetration. An extended immune response and booster effect
were also provoked indicating that the immune memory could be
obtained by this patch-vaccine. 95 Glenn et al, 96 demonstrated
that application of a patch containing heat labile enterotoxin (LT)
to humans has shown to produce robust LT-antibody responses.
These findings designated that TCI is feasible for human
immunization, and suggest that TCI may augment efficacy as
well as improve vaccine delivery. Similarly, it was shown by
Ishii et al, 97 that patch based TCI system could induce effective
immune responses to viral and bacterial infection. Recently patch
along with microneedles is used to provide non-invasive means
of vaccination and further has been demonstrated by more and
more experiments and clinical trials. 98,99
Second generation delivery systems (1990-2005)
Polymerosomes (PMs)
PMs were first generation delivery system developed in late
1990s. 100 They are self-assemble shells composed of amphi-
philic block copolymer, which consist of linked hydrophilic and
hydrophobic polymer chains and have amphiphilic properties
similar to phospholipids. 101 Polymerosomes have shown to be
stable in consideration of size and structure. 100 Based on the
selection of the block copolymer, its physical properties,
polymerosomes can be used as delivery systems with a wide
range of suitable properties. 102 Furthermore, polymerosomes
made up of degradable di-block polymer polyethylene glycol–
polybutadiene functionalized with an HIV-derived Tat peptide
effectively deliver the PMs to the DCs. 103 These findings
emphasize the potential exploit of polymerosomes as stout, virus
like antigen delivery systems.
Transferosomes
Transferosomes, also referred as a new class of tailored
liposomes, were first reported by Cevc and Blume 104 and are
described as deformable, elastic or ultra-flexible liposomes or
vesicles. 105 It is well known that barrier of stratum corneum
prevents hydrophilic and large molecular drugs through skin but
transferosomes are capable to constrict through area 1/10th the
width of the liposomes, permitting them to spontaneously
penetrate the stratum corneum. 106 Moreover, Cevc and
Blume, 104 reported that the osmotic gradient which is an
influential force for their diffusion into the skin was the created
by the difference in water amount of the comparatively
dehydrated skin surface and the hydrated viable epidermis.
Antigen entrapped in transferosomes induces stronger immune
response as compared to those entrapped in liposomes and
present in antigenic solution alone. 107 Mishra et al, 72 reported
that transferosomes loaded with hepatitis B surface antigen
(HBsAg) trigger enhanced antigen-specific systemic and muco-
sal antibody titer responses against HBsAg in vivo by
subcutaneous administration than other formulations including
mixture of transferosomes and HBsAg, HBsAg solution alone
and alum-adsorbed HBsAg administered intramuscularly. Epi-
cutaneous immunization of transferosomes loaded with gap
junction proteins transcutaneously shows higher permeation and
relative higher anti-GJP IgA in the serum than conventional
liposomes. 108
Ethosomes
Nowadays ethosomes have shown wide potential in TCI.
Safety concern and usefulness of ethosomes have been
influentially established as compared to other transcutaneous
carriers such as gels, patches and conventional liposomes. 109
They are vesicles composed of phospholipids, water and ethanol
with a concentration range up to 45%. 110 Although, the exact
mechanism for better permeation of the drugs into deeper layers
of the skin from ethosomes is still not clear. The synergistic
effects of two combinations are responsible for deeper
distribution as well as penetration in the lipid bilayers of
skin. 111 The promising physical distinctiveness of ethosomes is
their softness, flexibility and deformability. Rattanapak et al, 112
in their studies concluded that for TCI cubosomes and ethosomes
are promising lipid carriers in contrast to transferosomes and
liposomes. The first mechanism is the action of free ethanol
(ethanol effect). The polar head group of phospholipid present in
 M. Kaurav et al / Nanomedicine: Nanotechnology, Biology, and Medicine 12 (2016) 1063–1081
Figure 5. Schematic diagram of several transcutaneous antigen delivery technologies for TCI (A) patch, (B) viral vectors, (C) sonophoresis, (D)
electroporation, (E) vesicular systems such as liposomes, transferosomes, niosomes, cubosomes etc, (F) micro or nano solutions and emulsions, (G) tape
stripping, (H) microneedles, (I) permeability enhancers, (J) cell penetrating proteins.
SC disrupts by free ethanol that causes the structures changes
such as slackly disordered, increasing fluidity and membrane
permeability. Then ethosomal vesicles (ethosomal effect), which
are deformable and flexible straight forwardly infiltrate via the
disorganized SC inside deeper into the skin. A further predicted
mechanism is the fusion between ethosomes and skin lipids,
ensuing in active moiety release from the vesicles. The
effectiveness of ethosomes for topical vaccine delivery has
been explored. Mishra et al, 113 detailed that human DCs have
shown enhanced antigen uptake of HBsAg-loaded ethosomes
and the consequent triggering of an efficient Th1/Th2-type
immune response.
Immunostimulatory complexes (ISCOMs)
Liposomes have strong potential for immunostimulation and
their uptake by DCs is rapid, but now they have largely been
reinstated by the ISCOM formulations due to more stable
characteristics. ISCOMs are matrix constructs, incorporating
antigen, cholesterol and phospholipids with a 40 nm diameter, a
size comparable to that of many viruses. 114,115 Liposomes are
readily incorporated into the membranes of cells, may promote
endocytosis of antigen by DCs, monocytes and macrophages and
can enter the endosomal pathway for MHC-Class II presentation
with DCs, but not other cell types. Thus, they efficiently promote
T and B lymphoid cell activation. 116 Particles encapsulating
antigens with a 30-200 nm diameter such as ISCOMs 117 and
virus like particles (VLPs) usually also provide “threat” signals
that induce activation of APCs via TLRs. 118 Pandey et al, 119
prepared ISCOMs incorporating rHBsAg when administered
subcutaneously were shown to induce humoral as well as cellular
immunity and found to be immunogenic as compared to
marketed rHBsAg formulations by subcutaneous route of
administration. They also demonstrated potential of ISCOMs
as a carrier adjuvant for nasal subunit vaccines against hepatitis
1071
B for induction of humoral, cellular and mucosal immunity. 120
Their further research has shown that mucoadhesive ISCOMs
that retain in nasal cavity for longer duration of time may reduce
the dose/frequency for nasal vaccination. 121 Madsen and his
coworkers 122 investigated the effect of novel type ISCOMs on
compounds skin penetration and their potential as delivery
system for TCI.
Cubosomes
Cubosomes are of the nanometric colloidal dispersion of
lipids having self assembling property that makes bicontinuous
cuboidal liquid crystalline particulate structures, with unique
practical properties able to encapsulate amphiphilic, hydrophilic
and hydrophobic active medicaments. 123 They have distinctive
enclosing, carrying, solubilizing and shielding properties
because of large surface area and low viscosity, making them
an outstanding delivery system for various in vivo drug delivery
routes. 124 Boyd 125 detailed that simple diffusion force resulting
in rupture release of lipophilic drug from bulky cubic phases
depends on size and molecular weight of the drugs. Cubosomes
have also been reported to act as a valuable vaccine delivery
system to enhance efficiency and reduce boosting requirement
with improved IFN-γ production in animals vaccinated subcu-
taneously with ovalbumin and QA cubosomes as evaluated
against the control groups. 126 Bender et al, 127 envisaged about
skin penetration of SRB, a fluorescence hydrophilic model drug
encapsulated in cubosomes of mono-olein with the two-photon
microscopy and high accumulation fluroscence intensity in
micro-fissures and from which threadlike structure of fluores-
cence arrangement expands out laterally in skin tissues.
Vesosomes
Vesosomes are multicompartment structures which have
distinct inner compartments separated from the external
1072 M. Kaurav et al / Nanomedicine: Nanotechnology, Biology, and Medicine 12 (2016) 1063–1081

Table 4
Different generation delivery systems used for transcutaneous immunization.
Particulate Principle
systems
First generation delivery systems (before 90s)
Liposomes Inter membrane transfer,
adsorption
Fusion, phagocytosis
Nanoparticles Disruption of SC
by nano-bio interaction
with skin lipids
Niosomes Diffusion, interaction
with SC via fusion,
aggregation and adhesion
Patches Present high Ag protein
concentration gradient which
produce high driven force
for Ag penetration
Second generation delivery system (1999-2005)
Polymerosomes Internalization by
endocytosis, hydrolytic
degradation
Transferosomes Via diffusion
due to osmotic
gradient force
Ethosomes Fusion with cell
membrane proteins
ISCOMs Endocytosis by APCs
Cubosomes Simple diffusion
rapture release
Vesosomes Fusion with target cells
Third generation delivery system (2005 to present)
Gold NPs Via membrane interaction
and penetration without
membrane disruption
Properties Limitations Animal/effects Reference
67-71
Enhance activity against Biocompatible and Mice/induce IgG specific
extra and intracellular ecofriendly, low antibody, elicit Th1 and
pathogens, effective efficiency rapid clearance Th2 specific antibody response
immunoadjuvant from blood stream by
properties, enhance phagocytic cells
penetration
73-76,80,88
Seems alike natural pathogen in Can lead to particle Mice/increase uptake of
size and shape actively taken aggregation due to small antigen by LCs and increase
up by APCs improve antigen size and large surface level of IFN-γ and IL-2.
uptake and immunogenicity area, limited loading,
sometimes toxic
product form
89,90,92,94
Have characteristic size, shape May undergo fuse, Mice/elicit strong IgG
and fluidity, can entrap wide aggregate, leaking antibody titers, stimulate
range of molecules of entrapped serum immune responses
drugs, hydrolysis
95-97
Promotes Ag penetration Local irritation and Rat/induce mainly
across SC, induce Ag sensitization, Th2 type immune response
specific immune response contact dermatitis and antigen specific
immune memory
100-103
Self assemble shell, amphiphilic Low transfection efficiency, Mice/effective
properties make them suitable short-lived expression deliver antigen to DCs
carrier for both type molecules, of foreign gene
greater stability
104-107,72,108
Ultra flexibility, induce stronger Chemically unstable, Mice/robust mucosal and
immune response Compared inability to transport large systemic antibody response
to conventional liposomes molecules, expensive
109,111-113
Via increasing cell membrane Low diffusion Mice/enhanced
fluidity increase skin penetration, of Ag across SC Th1/Th2 cytokines
improved Ag entrapment efficiency level and uptake by DCs
114-116,119,122
Strong immuno-stimulation High cost Mice/upregulation of
potential, promote T and B Quil A toxicity humoral (IgG) as well as
cells activation cellular (IFN-γ and
IL-2) levels
124-127
Distinctive enclosing, Low loading efficiency Mice/higher IFN-γ
carrying, solubilizing, and less stability level and high penetration
shielding properties of peptide in skin
128-130,130
Have multiple Difficult to encapsulate Rat/significantly induce
compartment, can Large molecules serum IgG titer
encapsulate multiple and Th1 and Th2
material in same structure combined response
132,133,136,137
Huge surface area, high Cytotoxicity Mice/increased level
stability, easily fabricable of antigen specific
in different shape IgG and deep
penetration in skin layers

membrane. In a straightforward way it can be said as a larger
vesicle that deliberately encapsulates many smaller size vesicles
inside it. Each compartment of vesosome can encapsulate
different materials and have different bilayer composition. 128,129
Vesosomes (fusion liposomes) are novel carriers that can
competently support the intracellular delivery of encapsulated
antigen by fusion with target cells. The main drawback of
liposomes is that various vital drugs are released earlier than
favorable in vivo. 130 This problem is significantly addressed by
the vesosomes, while small molecules are released from
unilamellar liposomes in minutes, they are retained in vesosomes
from hours to days, even though the liposomes and vesosomes
have the same bilayer composition and size. 131 Mishra et al, 130
have demonstrated the fusion ability properties of phospholipid
liposomes made up of phosphatidylcholine dioleoyl phosphati-
dylethanolamine (DOPE), dioleoyl trimethyl ammonium pro-
pane (DOTAP) and dipalmitoyl phosphatidylcholine (DPPC) to
combine with principal phospholipid containing membrane
 M. Kaurav et al / Nanomedicine: Nanotechnology, Biology, and Medicine 12 (2016) 1063–1081
Figure 6. Schematic diagram showing generation of adaptive immune response following topical application of antigen containing NPs with or without adjuvant.
constituent. These types of fusogenic vesosomes have ability to
fuse with membrane of target cells and elicited strong immune
response after transcutaneous vaccination.
Third generation delivery systems (2005-present)
Inorganic nanoparticulate based vaccine-codelivery nanocar-
rier
Metal NPs such as quantum dots and iron oxide NPs are able
to penetrate through intact skin, of less than 10 nm in size, due to
its interaction with the lipid in extracellular space. Biocompatible
inorganic nanoparticulate based vaccine-codelivery is a new
choice that successfully evades the basic drawbacks of
conventional organic material carrier system in nanomedicine,
biomolecular and cellular applications, due to its inimitable
structural and compositional characteristics, for example huge
surface area, tunable surface characteristics by engineering
modifications and surface functionalization, plentiful physio-
chemical functionalities, high stability and explicit biological
nature. 132,133 Advancements in inorganic nanocarrier develop-
ment have made up our mind to focus on inorganic NPs for
controlled release specific targeting and control of their cellular
actions. 134 Yet much of work on inorganic NPs for TCI has not
been pursued. Elected applications of some useful inorganic NPs
1073
are reviewed in the next section and relevant review articles are
cited to provide further details.
Gold NPs (AuNPs)
Relevance of gold NPs (AuNPs) in biomedical and bioima-
ging constitutes a fast-growing field. 135 An interesting recent
report was revealed that superfine Au-NPs 5 nm were
investigated for TCI due to their skin permeable property.
Huang et al, 136 reported that when model protein drugs such as
horseradish peroxidase (HRP), β-galactosidase (β-gal) and
ovalbumin (OVA) were co-administered with Au-NPs (5 nm)
their dermal absorption increases and migrates into the deep skin
layers, which successfully enhanced the dermal absorption of
model proteins. It should be renowned that before application of
formulation skin hydration had been done for 10 min by covering
with the help of gauze to smoothen the process of uptake. In
another experiments cholera toxin (CT), a strong mucosal
adjuvant along with OVA was co-applied with the Au particles.
Normal saline solution of CT applied on back surface of shaved
mouse may play a role of an adjuvant for co-administered
vaccine and provoke high altitudes of CT-specific IgG
antibodies. Cholera toxin is also known for its leakable effect.
Au-NPs accumulated in high amounts in the stratum corneum
and in the epidermis. OVA along with CT in Au-NPs elicited the
production of IgG. 137 Recently, functionalized AuNPs have
1074 M. Kaurav et al / Nanomedicine: Nanotechnology, Biology, and Medicine 12 (2016) 1063–1081

Table 5
Recent clinical trial studies on transcutaneous immunization.
Title Clinical trial no. Clinical trial initiation and completion date Status Reference
181
ETEC Logical Trial (TREK) NCT00516659 May 2006/December 2007 Phase II (Completed)
182
Transcutaneous Immunization With An Attenuated NCT01311817 January 2011/July 2012 Phase I (Completed)
Listeria Monocytogenes Vector Vaccine
183
Safety Study of Recombinant Vaccine NCT01382095 July 2011/April 2013 Phase I (Completed)
to Prevent ETEC Diarrhea
184
Safety Study of Chimeric Vaccine to NCT01644565 August 2012/November 2016 Phase I (Ongoing)
Prevent ETEC Diarrhea (Expected completion)
185
Traveler's Diarrhea (TD) Automated Process NCT00751777 September 2008/November 2009 Phase II (Completed)
186
LT Vaccine Patch Self-Administration Study NCT00565461 November 2007/August 2008 Phase II (Completed)
187
Gender-Stratified Safety and Immunogenicity Study NCT01067781 February 2010/March 2012 Phase II (Completed)
188
Study of Allergen Immunotherapy in Grass NCT00777374 October 2008/November 2010 Phase II (Completed)
Pollen Allergic Subjects With Epicutaneous
Allergen (ZU-SkinSIT-003)
189
Safety Study Comparing a Vaccine NCT00261001 October 2005/September 2009 Phase I (Completed)
Transcutaneous Administration to
the Intramuscular Route (MANON-05)
190
Immunogenicity of Three NCT02075983 June 2013/December 2015 Phase I (Ongoing)
HIV GTU® Multi HIV DNA (Expected completion)
Immunizations Administered
Via Intramuscular, Intradermal
and Transcutaneous routes
(CUT‫٭‬HIVAC001)
191
Routes of Immunization And NCT01707602 October 2012/April 2013 Phase I/II (Completed)
Flu Immune Response
192
The safety immunogenicity of the DNA-GTU NCT02457689 May 2015/February 2017 Phase I/II (Completed)
Vaccine Administered to HIV-infected patients
on ART vs. Placebo (CUTHIVTHER001)
193
Vaccine Therapy and Resiquimod in Treating NCT00470379 April 2006/October 2012 Phase 0 (Completed)
Patients With Stage II, Stage III OR Stage
IV Melanoma That Has Been
completely removed by surgery
194
Immune response to yellow fever vaccination NCT00723489 August 2008/April 2011 Phase I/II (Completed)
in adults with atopic dermatitis
195
Study of safety and immunogenicity ISRCTN88861431 March 2004/March 2005 Phase I/II (Completed)
of Measles vaccine (Rouvax®)
administered by transcutaneous route

emerged as a delivery system for targeting, bioimaging and enhancer via different mechanism of action to overcome skin
biodistribution studies, 138 -141 biomolecules 142 or large size barrier and show synergistic effects action in TCI. Examples
biomolecules such as proteins, 143 DNA, 144 and RNA. 145 utilizing these approaches are below.

Synergistic enhancers/system combinations

Combination methods
Complex formulations
In previous text we had discussed about various delivery
systems, physical approaches and chemical approaches to
overcome the SC for TCI. But to be applicable in clinical use
almost it's a failure to achieve this goal with the use of single
approaches we discussed above. So it's a prerequisite to use
either combination of delivery systems together or delivery
systems with chemical approaches or with physical approaches
or all the three approaches together. They provide quite better
results in combination than they used alone. For example, when
two delivery systems used together for TCI suppose liposomes
with NPs, here liposomes containing lipid so they work to disrupt
SC and itself as adjuvant and NPs used as carrier system to
deliver vaccine material. Similar results have shown by chemical
It have been observed that the majority of the enhancers that
have been regarded as in the transdermal literature for their
ability to increase transport across skin have important
limitations such as not able to achieve the desired skin disruption
and cause skin irritation that is associated with their potency of
penetration. 146,147 Chemical mixtures of these enhancers or
synergistic combinations present several possible opportunities
to surmount the restrictions of single chemical enhancers. Many
examples of classical combinations are available such as
chemical mixture of two chemical enhancers, one of them
works on keratinocytes to open up transcellular and intracellular
pathways for infiltration of active moieties molecules and other
one acts on lipids of skin. Likewise, one component of mixture
combination of enhancer increases diffusion of therapeutic
molecules in stratum corneum and other one creates pathways for
them via disrupting. Some chemical enhancers in this
 M. Kaurav et al / Nanomedicine: Nanotechnology, Biology, and Medicine 12 (2016) 1063–1081
combinations are added to decrease or prevent degradation or to
enhance stabilization of therapeutics because they have proper-
ties of antioxidant, stabilizer and prevent enzymatic degradation
of proteins, peptides and vaccines. 148,149
Biphasic vesicles
Lipid based delivery systems which are composed of both
liposomes and emulsions are called biphasic systems. Biphasic
vesicle systems provide several advantages as a topical carrier
system such as having multichamber structure allows encapsu-
lation and co-encapsulation of various therapeutics of different
weight range with different solubilities. Biphasic systems are
evaluated for many vaccine candidates including proteins,
nucleotides, recombinant subunits and classical old inactivated
or killed vaccines and now became applicable to purposes in
human as well as in animals. 150 -152 Baca-Estrada et al, 150 have
shown topical delivery of hen egg lysozyme (HEL) and
leukotoxin antigens in biphasic delivery system induce strong
Th-2 cellular response and induce secretion of IgG1 antibody
response. Foldveri et al, 151 formulated biphasic delivery system
of plasmid DNA (pDNA) and have shown that it delivered
significant quantities of pDNA into the viable layers of human
skin in vitro and induced 5 times higher anti-IgD levels
compared with naked DNA alone and secreted IL-4 by cells
and induced cellular immune response. Babiuk et al, 152
demonstrated that topical delivery of plasmid in biphasic lipid
delivery system results to induce cellular and humoral response.
Intradermal application induces Th-1 whereas TCI induces Th-2
type cellular immune response.
Micro and nanoemulsions
Micro and nanoemulsions is colloidal carrier system that
offers several advantages for drug delivery, such as ease of
preparation, long-standing stability, soaring solubilization ca-
pacity for hydrophilic and lipophilic agents. 153 Microemulsion
has shown great prospective in absorption and delivery
enhancement of peptides and pDNA, especially the water-in-oil
(W/O) type, 154 and shown enhanced penetration properties in
stratum corneum due to their lipophilic nature. 155 Although it
has not been widely reported yet for the application in
transcutaneous vaccine, nanoemulsion has showed its advantage
in topical delivery of hydrophobic drugs due to their lipophilic
nature. Nanoemulsions are isotropic novel drug delivery systems
consisting of two liquid phases immiscible with each other,
emulsified oil and water systems with mean nanometric droplet
size range. They are either oil-in-water (o/w) or water-in-oil (w/
o) type systems, where the internal phase of the particle is either
oil or water, respectively. Nanoemulsion containing antigen and
adjuvants effectively taken up by APCs efficiently induces
cellular as well as humoral immunity and increases secretion of
IgG and IgA antibodies in serum. So emergent mucosal
immunity via use of nanoemulsion may turn out to be an
important tool in the future struggle against HIV. 156 In some
cases, nanoemulsion works as adjuvant for vaccine formulations
applied to the mucosal surface and it helps uptake by APCs. This
results in the significant systemic and mucosal immune response
due to that the production of specific IgG and IgA antibody as
1075
157 158
well as cellular immunity. Makidon et al, who prepared
nanoemulsion adjuvant system of rHBsAg for nasal vaccination
demonstrated that this vaccine induces a Th1 associated cellular
immunity. Supplementary research has been ongoing in animal
trials for other vaccines including anthrax and hepatitis B. This
nanoemulsion technology has been licensed by NanoBio
Corporation for vaccine development. 159
Other formulations
Other than above mentioned methods and delivery systems,
more new approaches to overcome problems associated with TCI
have been discussed here. Su et al, 160 reported about
hydrolytically degradable loaded protein and oligonucleotide
was incorporated into a novel multilayer film for the transcu-
taneous delivery. Results showed OVA loaded from LbL films
penetrated into barrier-disrupted skin was uptaken by skin APCs
and transported to the skin-draining lymph nodes. Tahara et
al, 161 successfully achieved TCI by applying a solid-in-oil (S/O)
nanodispersion, without the use of any skin pre-treatment or
adjuvant. S/O of hydrophilic drugs has effectively enhanced the
permeation of proteins into the skin. Result outcomes have
shown that upon incorporation of low molecular drug in elastic
vesicles enhanced their transdermal diffusion through the skin
and vesicular structures are reached in the deep layer of stratum
corneum. In another report about, nanofiber merged, an
uninterrupted, nonwoven nanofibrous membrane containing
nanoencapsulated protective antigen (PA) is produced through
the process of electrospinning. This novel formulation provides
the basis for the transdermal anthrax vaccine. 162
Devices and formulations
As we previously discussed in this review removal or
disruption of SC increases the transcutaneous permeability for
antigens and makes them effectively available for captured by
APCs present in skin layers. Many studies also revealed that
transcutaneous application of formulation system alone was not
able to induce effective immune responses. To make these
approaches possible physical methods and chemical enhancers
along with novel vaccine delivery systems are used. Here below
we shortly discussed about this concept with few examples.
Chemical enhancers with delivery systems
Synergistic combination possible for vesicular colloidal
delivery is made up of concentric bilayers of self-assembled
amphiphilic molecules. Vesicular systems have achieved
eminence as skin penetration improving agents as well as
protein/vaccine carrier agents in transdermal delivery. 69,163 The
physiochemical characteristics of chemical enhancers have an
intense outcome on the deeds of the vesicles and therefore on
their efficiency in enhancing vaccines and therapeutics delivery
through skin. 104,112,163-169 Synergistic relations between the
components of the vesicular system and between the different
vesicular systems and skin elements are alleged to be
accountable for the advanced skin permeation enhancement
through vesicular systems.
1076 M. Kaurav et al / Nanomedicine: Nanotechnology, Biology, and Medicine 12 (2016) 1063–1081
Physical methods with delivery systems
Various physical methods are available to increase TCI
potential such as microneedle techniques, ultrasound, electropo-
ration, laser, hyperthermia technique, gene gun and tape
stripping etc. microneedle array mostly prepared along with
patch formulations and being delivered to the skin in painlessly
manner. Alarcon et al, 170 reported that influenza vaccine
administration along with microneedle induces higher immune
response than that of intramuscular injection in animal model.
Koutsonanos et al via metal microneedle deliver inactivated
influenza lethal virus and induce strong immune response. 171
Similar finding for immunization by vaccine coated micro-
needles is shown in several studies. 98,99,171,172 Tape stripping
and degreasing with chemical promote dermal immunization via
skin disorganization, partial leaching of lipids and induce APCs
maturation and migration. 173,174 Huang et al, 175 using low
pressure gene gun deliver NPs transcutaneously and induce
strong Th2 immune response. Electroporation high voltage pulse
techniques cause structural perturbation of skin lipid bilayer and
are widely used in TCI. 176 Low frequency ultrasound techniques
increase antibody titer because of their adjuvant action and are
used in delivery of vaccine delivery systems through induced
inflammation. 177,178 Thermal hyperthermia through activation
and migration of LCs and because not causing any pain and
damage to skin should be promoted as a unique technique for
TCI. 179,180 All the above examples have shown that physical
method along with delivery system came to be beneficial for
improved TCI.
Clinical trials of transcutaneous immunization
There are several numbers of diseases such as tetanus,
measles, mumps, pertussis, rubella, diphtheria, Haemophilus
influenzae type b (Hib), yellow fever and polio which are from
very last decade under command because of vaccine creation.
The attention on the development of vaccine becomes more
attractive due to the wide use of TCI. Several preclinical trials of
transcutaneous immunization have been reported and some of
them successfully reached the clinical trial stage. Clinical trials
reported on TCI in recent few years are summarized in Table 5.
Conclusions and future prospective
The prime motive of vaccine development is the establish-
ment of manufacturing technologies that provide secure and
effectual vaccine antigen in haste and soundly, but this problem
to bear out as much as necessary vaccination to prevent
infectious diseases stays behind to be solved. It is now a
well-known fact that the skin is an attractive immunological
avenue that offers non-invasive, needle free, painless, effective,
easy-to-use, low cost prophylaxis against infectious diseases, and
better patient compliance with few side effects and secured
handling than conventional injections. The main challenges to be
pointed in transcutaneous vaccination are to make certain precise
delivery into the epidermal or dermal skin of antigens through
subcutaneous where LCs and DCs exist and to formulate antigen
with adjuvant and/or carrier system for effectual activation of
defined type of cells/receptors/ligands that exists in DC subsets.
Many diverse advances have been enlarged of which quite a lot
of methods may escort to flourishing TCI by combining the
novel formulation approaches with physical/chemical ap-
proaches together with addition of an adjuvant that activates
specific receptor for robust immune response which we have
discussed in our section approaches to improve TCI. As we
discussed in this review, various approaches to disrupt stratum
corneum and formulations have been developed, further, basic,
clinical and preclinical studies have also been conducted, but still
most of transcutaneous vaccines are in preclinical or clinical trial
stage just because the most efficient way toward this is at a halt
and desires to be established, which will necessitate cooperative
endeavors of pharmaceutical experts, vaccine development
experts, immune experts and mechanical engineer experts.
Only then TCI can effectively improve and in the near future
revolutionize vaccination method which would contribute to a
global countermeasure against infectious diseases and would
provide many countries with cost effective and beneficial
vaccination.
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