International Immunopharmacology 11 (2011) 1516–1522

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International Immunopharmacology

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Non-ionic surfactant vesicles mediated transcutaneous immunizationagainst hepatitis B

Chetan Maheshwari a,⁎, R.S. Pandey a, Akash Chaurasiya b, Ashu Kumar c, D.T. Selvam c,G.B.K.S. Prasad d, V.K. Dixit a

a Department of Pharmaceutical Sciences, Dr. Hari Singh Gour University, Sagar 470003, Indiab Faculty of Pharmacy, Jamia Hamdard, New Delhi-110062, Indiac Defence Research and Development Establishment, Gwalior 474002, Indiad Jiwaji Vishwavidyalaya, Gwalior, India

⁎ Corresponding author. Tel.: +91 8010110041.E-mail address: chetansemail@gmail.com (C. Mahes

1567-5769/$ – see front matter © 2011 Elsevier B.V. Adoi:10.1016/j.intimp.2011.05.007

a b s t r a c t

a r t i c l e i n f o

Article history:Received 22 December 2010Received in revised form 20 April 2011Accepted 9 May 2011Available online 24 May 2011

Keywords:NiosomesTranscutaneous immunizationHBsAg surface antigenCholera toxin B subunitConfocal laser scanning microscopy

Transcutaneous immunization (TI) has many practical merits compared to parenteral routes of administra-tion. In the present study, non ionic surfactant vesicular carrier, i.e. niosomes, was evaluated for topicaldelivery of vaccines using hepatitis B surface protein as an antigen and cholera toxin B as an adjuvant.Niosomes were characterized for size, shape, entrapment efficiency and in process antigen stability. In vitropermeation and skin deposition studies of antigen were performed using human cadaver skin. Skinpenetration efficiency of niosomes was assessed by confocal laser scanning microscopy. The immunestimulating activity of these vesicles was studied by measuring the serum IgG titer, isotype ratio IgG2a/IgG1and mucosal immune responses following transcutaneous immunization in Balb/c mice and results werecompared with the alum adsorbed HBsAg given intramuscularly and topically administered plain HBsAgsolution. The result shows that optimal niosomal formulation could entrap 58.11±0.71 of antigen withvesicle size range of 2.83±0.29 μm. Serum IgG titers after three consecutive topical administrations weresignificantly better than single administration of hepatitis antigen with niosomal system, suggesting aneffective stimulation of serum immune response; higher IgG1/IgG2a ratio revealed CTB mixed niosomes elicitboth Th1 and Th2 responses. This study suggests that topical immunization with cholera toxin B is potentialadjuvant for cutaneous immune responses when coadministered with the HBsAg encapsulated niosomes.Results also suggest that the investigated niosomes systems can be effective as topical delivery of vaccines.

hwari).

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© 2011 Elsevier B.V. All rights reserved.

1. Introduction

Transcutaneous immunization (TI) is a novel vaccination strategybased on the application of antigen together with an adjuvant ontohydrated bare skin and subsequent delivery to underlying Langerhanscells that serve as antigen-presenting cells [1]. Non-invasive vaccinationonto the skin could improve vaccination programsbecause the proceduredoesnot require specially trainedpersonnel andmayavoid riskassociatedwith needle prick. A critical role in enhancing the immune response toboth licensedandexperimental vaccines isplayedbyadjuvants. It appearsthat adjuvants are crucial to inducing sufficiently potent and functionalimmune responses on the skin. The uses of adjuvants have recast thefields of vaccine research for injectable, oral, and nasal immunization.Adjuvants, such as cholera toxin and heat labile enterotoxin fromEscherichia coli and other adjuvants, and delivery techniques that cantarget LCs may revolutionize the future delivery of vaccines [2,3].

One of the possibilities for increasing the penetration of bioactivesthrough the stratum corneum is the use of vesicular systems.Interactions can occur either at the skin surface or in the deeperlayers of the stratum corneum. Previously it has been reported thatadsorption and fusion of vesicles onto the skin surface result in theformation of lamellae and rough structures on top of the outermostcorneocytes [4,5]. These delivery systems act as adjuvants to enhancethe immunogenicity of antigens, which otherwise induce “weak”immune response when applied topically. Nonionic surfactant basedvesicles (niosomes) that are assemblages of non-ionic amphiphilesinto closed bilayer structures have also been reported to possessstrong adjuvanticity [6]. Vesicular carrier systems liposomes andniosomes have been advocated for topical delivery of bioactives [7–9].The low cost, high purity, content uniformity, greater stability andease of storage have presented niosomes as better alternatives toliposomes. Cholera toxin (CT) and its non toxic components choleratoxin B (CTB) have been important in the development of the mucosalroute as a useful and easily accessible non-invasive way to induceimmunity [10–13]. Langerhans cells (LCs) are found in the epidermis,almost directly under the stratum corneum. Their superficial location

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makes these potent APCs attractive targets for vaccine delivery. LCs isthought to be phagocytes antigen in the skin and, if activated, willmigrate to the draining lymph nodes where they present antigen toT cells. CT activates LCs, which leads to antigen presentation in thedraining lymph node that leads to the induction of a systemic immuneresponse [14]. Employing CT as an adjuvant for transcutaneousimmunization would allow the skin to be used in a similar way.

The aim of the present studywas to establish potential of niosomesas topical hepatitis B vaccine carriers. Niosomes encapsulatinghepatitis B surface antigen (HBsAg) were prepared and characterizedby variety of analytical methods. The specific immunological responseagainst hepatitis B antigen elicited by niosomes was compared withthat induced by administration of HBsAg vaccine through topicalroute and intramuscular administration.

2. Materials and methods

2.1. Materials

The HBsAg and CTB were obtained as gift samples from ShanthaBiotechnics (Hyderabad, India) and Institute ofGenomics and IntegrativeBiology, New Delhi respectively. AUSZYME monoclonal diagnostic kitwas obtained from Abbott Laboratories, Chicago, IL, USA. Cholesterol,soyaphosphatidylcholine (SPC), SephadexG-100, tetramethyl benzidine(TMB), FITC-OVA, FITC-dextran (MW=21 kDa), horseradish peroxidase(HRP) labeled goat anti-mouse IgG, IgG2a and IgG1 antibodies werepurchased from Sigma (Sigma, St. Louis, USA). Span 80 (an esterof plain sorbitan with fatty acids) was purchased from Himedia(Mumbai, India). Gel electrophoresis kit was purchased from theGenei (Genei Pvt. Ltd., Bangalore, India) and used for SDS–PAGEstudies. All other chemicals and reagents were of analytical grade,purchased from local suppliers and used as received. Distilleddeionized water (Milli-QTM Water system, Millipore Corporation,Massachusetts, and USA) was used throughout the study.

2.2. Preparation of vesicular carrier systems

Niosomes were prepared by reverse phase evaporation method[15,16]. Briefly, Span 80 and cholesterol in a molar ratio 7:3 weredissolved in diethyl ether followed by emulsificationwith 2 ml aqueoussolution of HBsAg solution (10–150 μg/ml) by probe sonication(Soniweld, India) for 2 min at 50 Kc/s. Thick emulsion was formedwhichwas thenkeptover a vortexmixer inorder to remove any residualether. To this emulsion, 3 ml phosphate buffer (PB, pH 6.5)was added inorder to hydrate the vesicles.

2.3. Entrapment efficiency

For determination of entrapment efficiency the unentrappedantigen from niosomal formulation was separated by the use of theSephadex G-100minicolumn centrifugationmethod [17]. The amountof antigen entrapped in the vesicles was then determined bydisrupting the vesicles using 0.1% Triton X-100 followed by filtrationusing 0.22 μm membrane filter (Millipore, USA). The amount ofantigen was determined by biscinchoninic acid (BCA) protein assaymethod for HBsAg by taking bovine serum albumin as standard.

2.4. Vesicle morphology and size analysis

Prepared vesicular systems were characterized for their shapeusing transmission electron microscopy (TEM) with an acceleratingvoltage of 100 kV. A drop of the sample was placed on to a carbon-coated copper grid to leave a thin film. Before the film dried on thegrid, it was negatively stained with 1% phosphotungstic acid (PTA)and excess of the solution was drained off with a filter paper. The gridwas allowed to thoroughly dry in air and samples were viewed in

a transmission electron microscope (Phillips Morgagni D-268,Netherlands). For the size measurement, vesicular suspension wasmixed with the appropriate medium (PB pH 6.5) and the sizemeasurement was done by Malvern Zetasizer 3000 HS (MalvernInstruments Co., UK). Each experiment was conducted in triplicate.

2.5. In process stability

SDS–PAGE experiment was carried out to analyze the integrity ofHBsAg antigen under non-denaturing condition. In presence of strongreducing agent and heat, proteins get dissociated before they wereapplied on the gel. SDS–PAGE experiments were performed on a 5%stackinggel and8%resolvinggel following thestandardprotocol. Thegelwasdriedand the sampleswere appliedon to thegel for electrophoresis.Protein bands were detected by Coomassie blue staining. The sampleswere heated to 95 °C for 5 min prior to their applications.

2.6. In vitro skin permeation and deposition studies

The in vitro skin permeation of rHBsAg-loaded niosomes and CTB,plain niosomes, plain rHBsAg solution and CTB, was studied usinglocally fabricated Franz diffusion cell with an effective permeationarea of 2.8 cm2. The temperature was maintained at 32±1 °C. Thereceptor compartment contained 7 ml PBS (pH 6.5) and wasconstantly stirred by magnetic stirrer (Expo India Ltd., Mumbai,India) at 100 rpm. Dermatomed (~500 μm thickness) human cadaverskin from abdominal areas was obtained from District Hospital, Sagar,India, and stored at −20 °C. The skin was then carefully checkedthrough a magnifying glass to ensure that samples were free from anysurface irregularity such as tiny holes or cervices in the portion thatwas used for transdermal permeation studies. After assurance, theskin was mounted on a receptor compartment with the stratumcorneum side facing upward into the donor compartment. Formula-tions containing 10 μg of HBsAg were applied on the skin in donorcompartment. Samples were withdrawn through the sampling portof the diffusion cell at predetermined time intervals over 24 h andanalyzed. The receptor phase was immediately replenished withequal volume of fresh diffusion buffer. After dismantling wascompleted, the donor compartment of the cell was rinsed carefullyfive times with 0.5 ml of buffer solution. The skin was removed andwashed with 0.5 ml of buffer solution.

Approximately five such washings were found to be sufficient toremove N99% of the formulation when determined at time zero. Allwashings were collected and assayed for hepatitis B. Following therinsing procedure, the skin patch was mounted on a board and a pieceof surgical adhesive tape (1.9 cm wide and about 6 cm long) waspressed firmly to the skin surface with a spatula. The tape was ofsufficient size to cover the area of skin that was in contact with theformulation. Twelve such strippings were carried out, and each stripwas analyzed separately for HBsAg.

For the HBsAg analysis of the donor compartment washings, skinwashings, and receiver compartment solution, enough trichloroaceticacid (10% w/v) was added to samples so that the concentration of theprotein precipitant was ~5% (w/v). After overnight equilibration, themixture was centrifuged (Remi C-24, Mumbai, India) for 15 min3000 rpm and the supernatants and precipitates were separatelyassayed. For HBsAg analysis of the skin samples (tape strippings andremaining skin), a 10% (w/v) solution of trichloroacetic acid in HEPESbuffer was added to the sample contained in a flask and the contentswere thoroughly mixed on a Vortex mixer at 1200 rpm (FischerScientific, India) and further incubated for 24 h to allow intimatecontact between the protein precipitant and the HBsAg residing in theskin tissue. The mixture was centrifuged for 30 min (3000g) and thesupernatant and precipitate were assayed. The quantitative estimationof antigenswas performed according to themanufacturer's instructions

Table 1Immunization protocol for HBsAg formulations (n=5).

Transcutaneous immunization⁎

Group1

10 μg HBsAg in PBS pH 7.4 Priming (at day 0, 1 and 3 consecutivedays) and one booster doses on 14 days

Group2

Niosome and HBsAg physicalmixture

Group3

HBsAg, CTB and niosomesphysical mixture

Group4

HBsAg loaded niosomes andCTB physical mixture

Intramuscular immunizationGroup5

2 μg alum adsorbed HBsAgformulation (positive control)

One priming and one booster doseafter 3 weeks

⁎ Total volume of formulation was kept constant to 50 μl. HBsAg and CTB quantitywere fixed at 10 μg and 50 μg respectively.

Table 2Characteristics of prepared niosomes.

S.No.

Parameters After preparation After storage for3 months at 5 °C

Plainniosomes

HBsAg loadedniosomes

Plainniosomes

HBsAg loadedniosomes

1 Mean particlediameter (μm)

2.25±0.26 2.83±0.29 2.57±0.39 3.21±0.56

2 Polydispersityindex

0.145±0.02 0.163±0.05 0.167±0.02 1.88±0.08

3 Encapsulationefficiency (%)a

– 58.11±0.71 – 56.32±1.1

All values are mean±SEM (n=3).The results are shown as mean value (±upper/lower value of duplicate means).

a Relative content (relative to that of initial antigen) antigen, inactivated sandwichELISA divided by protein content.

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(AUSZYME assay kit, Abbott Laboratories, Abbott Park, IL). Triplicateexperiments were conducted for each study.

2.7. Immunization experiments

Female Balb/c mice (age 7–9 weeks; weight 15–20 g) were usedfor in vivo evaluation of prepared formulations and each groupcomprised of five animals. The study protocol including handling, careand immunization was approved by Institutional Animal EthicsCommittee of Dr. Hari Singh Gour University, Sagar, India. The studywas carried out in accordance with the guidelines issued by theCommittee for the Purpose of Control and Supervision of Experimentson Animals (CPCSEA, Ministry of Culture, and Government of India).For topical application of formulations, animal hairs were carefullyshaved on dorsum. The skin was then carefully wiped with 70%ethanol; this removes oil and dry epidermal cells. For topicalimmunization, 200 μl of immunizing formulation (10 μg equivalentdoses) was placed on the shaved skin over a 2 cm2 area and left to dry.No adverse effects from the shaving, anesthesia, immunization, orwashing procedures were generally observed. Animals were primarilyinoculated with the topically administered niosomes formulations onthree consecutive days and followed by topical booster dose on day21st day (Table 1). Immune response was also compared with alum-adsorbed HBsAg (10 μg) given intramuscularly.

Fig. 1. TEM photograph of niosomal formulation.

2.7.1. Collection of samplesPre-immune samples of serum were obtained on day 0 before

immunization. Subsequent to immunization, collections were made atdays 21 and 42. Serumwas obtained by centrifugation of blood samplescollected from retro orbital plexus of mice under ether anesthesia.

2.7.2. Antibody detectionAntibody responses in the serum of immunized animals were

determined using a microplate ELISA. Microtiter plates (Nunc-Immuno Plate® Fb 96 Mexisorp, NUNC) were coated with 100 μl/well of 10 μg/ml HBsAg antigen in PBS (pH 7.4) and incubatedovernight at 4 °C. The plates were thoroughly washed with PBS-Tween 20 (PBS-T, 0.05%, v/v). The serum samples were serially diluted1:1 with PBS and 100 μl of each sample was added to each well ofcoated ELISA plates. The plates were incubated for 1 h at roomtemperature and washed three times with PBS-T. 100 μl of peroxidaselabeled goat anti-mouse IgG, IgG2a or IgG1 was added to each well.The plates were covered after incubation for 1 h at room temperature.Washing was repeated. 100 μl of tetra methyl benzidine solution wasadded to eachwell followed by addition of 50 μl of H2SO4 after 90 min.After 15 min the intensity of developed color wasmeasured at 450 nmusing a microplate reader (Bio-Rad, USA). The OD measured isproportional to the amount of HBsAg in the known samples. TheHBsAg content is calculated using a parallel line analysis [18]. PlainHBsAg (provided by Shantha Biotechnics Ltd., Hyderabad, India) wasused as a control standard sample in the same concentrations.

2.8. Confocal laser scanning microscopy

Depth and mechanism of skin penetration of macromolecularfluorescent marker FITC-dextran loaded niosomes were investigated

Fig. 2. Particle size distribution of niosomal formulation.

Fig. 3. SDS–PAGE electrophoresis (a) pure HBsAg; (b) HBsAg extracted from niosomal formulations.

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using confocal laser scanning microscopy (CLSM). FITC-dextranencapsulated vesicular carriers, i.e. niosomes and plain FITC-dextransolution, were applied topically to the shaved skin of mice. After 4 hthe mice were killed and the skin was removed. Microtome wasperformed and ribbons of sections (thickness 6 μm) were fixed ontoglass slides using egg albumin as the fixative. The sections wereviewed under a confocal laser scanning microscope (Zeiss CLSM 501equipped with Zeiss Neofluor 40/1.3 objective and Zeiss LSM 510software, Jena, Germany).

Table 3Distribution of HBsAg in various strata of the skin 24 h after in vitro topical applicationof HBsAg-containing formulations to full-thickness skin.

Formulation Distributiona on or in:

Stratumcorneumsurface

Deeperstratumcorneum

Deeperskin

Combined donorcells and skinwashing

HBsAg plain (in PBS pH 7.4) 8.1±2.6 6.3±1.9 0.4±0.3 75.6±11.3HBsAg and niosomalphysical mixture

42.3±5.1 31.1±4.6 2.2±1.2 14.2±3.5

HBsAg loaded niosomes 6.7±2.8 48.9±7.4 8.1±5.1 21.4±4.1

a Expressed as a percentage (±standard deviation) of the amount of formulationapplied (n=3 to 5).

3. Result and discussion

3.1. Preparation and characterization of niosomes

Topical immunization using CTB adjuvant based vaccines is anappealing strategy, where extrinsic antigens can be expressed in viablecells of skin associated lymphoid tissues (SALT) in sufficient quantitiesfor eliciting immunological response. Moreover, vaccine can beadministered in a more patient friendly and non-invasive manner. Inthis study, non-ionic surfactant based vesicles (niosomes) weredeveloped for efficient delivery of HBsAg for topical immunization.Niosomes were prepared by reverse phase evaporation techniquebecause the highest skin penetrating and bioactive carrying capabilityis frequently associatedwith suchmethod [15,19]. Further, this methodis also associated with relatively higher encapsulation efficiency ofmacromolecules such as antigens and peptides. Percent entrapmentefficiency obtained was 58.11±0.71% for niosomes. Themorphology ofniosomes was examined with a transmission electron microscope. Thevesicle appears dark with the bright surroundings. TEM photographs(Fig. 1) further confirmed that the vesicles were spherical in shape.Mean vesicle size of niosomes was found to be 2.83±0.29 μm withpolydispersity index 0.145 (Table 2 and Fig. 2).

The physical stability of the niosomes after storage at 5 °C for3 monthswas studied by DLS. The size and polydispersity of these lipid-based subunit vaccines had not changed significantly (Table 2). Thisfinding implies good physical stability of these vesicular systems. In-

process stability of the encapsulated HBsAg was assessed using SDS–PAGE electrophoresis. It is widely used for analysis and characterizationof protein samples. The gel was run with spots of pure HBsAg, HBsAgextracted from vesicular formulations, and only a single visible band forpure aswell as extracted HBsAgwas observed in the gel at the same loci(Fig. 3a, b). This reveals that the preparation conditions did not causeany degradation of HBsAg antigen.

3.2. In vitro skin permeation and deposition studies

In-vitro skin permeation experiment was investigated by Franz-diffusion cell. Permeation of antigen across mice skin via differentformulations revealed better deep skin deposition of HBsAg-loaded inniosomes formulation as compared to other formulations (pN0.05).The percentage cumulative permeation of HBsAg in various strata of theskinniosomes andCTB, plain niosomes, plain antigen solutionandCTB isreported in Table 3. The effect of vesicle on stratum corneum maychange the bioactive permeation kinetics due to an impaired barrierfunction of the stratum corneum. These results are in accordance withthe previous findings which demonstrate better skin permeabilityof drug through topical niosomal formulations [8]. Surfactants in

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formulations serve as penetration enhancer by raising the fluidity andreducing the barrier property of stratum corneum [20,21].

3.3. Confocal laser scanning microscopy

Confocal laser microscopy was utilized to confirm the permeationof macromolecular fluorescent marker, i.e. FITC dextran containingvesicular constructs onto the mice skin. CLSM photomicrograph

Fig. 4. CLSM photomicrograph (a) FITC dextran nioso

revealed deposition of FITC dextran loaded vesicular carrier constructsin different layer of skin through lateral diffusion (Fig. 4a). It wasobserved that the distribution of fluorescence intensity at differentdepths in the skin demonstrated that the niosomes associated dyewas transported between and along the lipid stacks in the inter-cellular space. The intensity of fluorescence was diminished with thedepth in dermis; higher fluorescence in the epidermal skin tissues incase of niosomes systems may be attributed to the presence of deeper

mal formulation; (b) plain FITC-dextran solution.

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skin penetration of niosomes system that might favor betterlocalization of the vesicles into skin compartment. The fluorescenceintensity gradually decreased with the depth in the skin. This may beattributed to the fact that niosomes containing dye were facing sinkconditions below the stratum corneum. As a result, dye dilution andfinally its elimination through the lymphatic drainage system occurred.Plain FITC-dextran solution was not found to penetrate into deeperskin layers; the fluorescence in this case was mainly confined to thesuperficial skin layers (Fig. 4b).

3.4. Immunological response

We examined the nontoxic cell-binding B subunit (CTB) as apotential adjuvant for cutaneous immune responses when coadmi-nistered with HBsAg encapsulate niosomes. The level of anti-HBsAgantibodies (IgG) was determined for all experimental groups after 21and 42 days. Table 4 shows serum IgG response, obtained with threeinoculations in consecutive days and intramuscularly primed alumadsorbed antigen respectively. Booster immunization was performedfor all groups after 3 weeks.

After priming the HBsAg loaded niosomes and CTB administeredtranscutaneously and was able to elicit strong anti-rHBsAg IgGantibodies while HBsAg PBS induced very low antibodies titer.Furthermore, an enhanced responsewas also observed after the boosterinjection,where in group 4 responses were increased to HBsAg bymorethan 120-fold (pb0.001) (Table 2) as compared to HBsAg in PBS. Afterthe boost, HBsAg loaded niosomes with CTB elicited 8-fold higherantibody response compared with the primary response, indicatinginduction of memory cells after priming. Balb/c mice immunized withdeveloped higher niosomal IgG antibody titers than mice receivingHBsAg alum (six foldmore in primary response (pb0.001) and two foldmore in secondary response (pb0.01)). Results obtained from IgG1 andIgG2a anti-rHBsAg titers determined by ELISA are consistent withprevious data demonstrating that alum induces a Th2 response,characterized by very low IgG2a responses [22,23]. UndetectableIgG2a primary or secondary response was elicited by the HBsAg alone,while a weak IgG2a antibody titer was elicited by HBsAg alum in thesecondary response only. In contrast, mice injected with HBsAg loadedniosomes with CTB produced high levels of IgG2a. The consistentlyhigher IgG2a anti-rHBsAg titers obtained, in both primary (pb0.001)and secondary (pb0.02) responses, in mice treated with CTB mixedHBsAg loaded niosomes than in mice treated with HBsAg alum wasobserved in all mice. The CTB mixed HBsAg loaded niosomes elicitedthe highest IgG1 anti-rHBsAg titers in both primary (pb0.001) andsecondary (pb0.01) responses. Alum produced high IgG1 titers mainlyafter the boost, although to a lesser extent than niosomes (pb0.01).Because IgG1 is driven by IL-4 (Th2), and IgG2a is driven by IFN-γ (Th1)an increase of IgG1/IgG2a ratio after vaccination indicates a Th2response, and a decrease in this ratio after immunization indicates aTh1 response. Alum induces almost only a Th2 response, while CTB

Table 4Primary and secondary immune responses against rHBsAg formulations in Balb/c mice.

Groups Antibody titers

21 Days after priming

IgG1 IgG2a IgG

Group 1 b1 ND b1Group 2 1.12±0.02ab b1 1.84±0.11a

Group 3 1.34±0.06 b1 1.91±0.03Group 4 2.14±0.31 1.23±0.25 3.24±0.47Group 5 3.04±0.40a b1 3.05±0.36a

Antibody titers are expressed as log mIU/ml (mean±SD). ND: not detectable. Groups of 6 Baat 3-week intervals associated with niosomes (10 μg). 1 μg of HBsAg either in PBS or alumStatics: a(pb0.001) significant against the group of mice immunized with HBsAg alonesignificant against the group of mice immunized with the alum vaccine according to Tukey

mixed niosomes elicits both Th1 and Th2 responses as demonstrated bythe strong IgG2a and IgG1 anti-rHBsAg antibody responses. Theseresults suggest that both adjuvants modulate differentially the type ofimmune response to immunization with rHBsAg.

The exceptional adjuvant activity CTB on cell-mediated immuneresponses to antigen co applied onto the skin appears to be in partassociatedwith their capacity to induce the rapid recruitment of DC intothe exposed epidermis and their subsequent exit from the epitheliumto the draining LNs. In this respect, exposure of the skin to CTB wasassociated with a rapid increase in DC density in the epidermis andparticularly in the underlying dermis [24].

It was observed that formulations are capable of eliciting acombined serum IgG2a/IgG1 response. Different formulations loadedwith antigen could be differently processed and presented to aspecific T helper lymphocyte subpopulation, which could originatedifferences in the specific serum IgG2a/IgG1 antibody production.These results are in accordance with the findings of Banchereau et al.[25]. They suggested that particulate antigens can be processed andpresented either by MHC class I or MHC class II by dendritic cells andmacrophages, which stimulates Th1 and Th2 lymphocyte subpopula-tions. This reflects that topical administration of HBsAg encapsulatedniosomes, coadministered with CTB can elicit both Th1 and Th2immune response; however more studies concerning cell mediated(T-cell) proliferative assays and interleukin production should beconducted in order to completely characterize the immune responseelicited by this system.

Various mechanisms have been established for topical delivery ofbioactives via liposomes or niosomes. Vesicle composition and henceits physico-chemical properties demonstrated consistently that thephases of the amphiphiles formed in the membranes of the vesicles(a liquid or a gel phase) are an important feature, which plays a vitalrole in its effectiveness as a (trans)dermal delivery vehicle. Perme-ation studies in vitro have revealed that liquid-state vesicles are moreeffective than gel-state vesicles in enhancing drug transport [26,27].Vesicle adsorption and fusion onto the surface of skin lead to a highthermodynamic activity gradient of bioactive-stratum corneuminterface [7]. The vesicles in addition to their inherent ability to bebetter taken up by the APCs also protect antigen from degradation byenzyme attack. Moreover, vesicles could also act as rate-limitingmembrane barrier and serve as a local depot for the sustained releaseof encapsulated HBsAg antigen [7]. Thesemight be the possible reasonfor a well-sustained titer value using the vesicular carriers.

Surfactants in formulations serve as penetration enhancer byenhancing the fluidity and reducing the barrier property of stratumcorneum [19,20,28]. Moreover, the principle constitutive lipid ofniosomes (span 85) is composed of unsaturated fatty acids (trioleatechains) and the presence of unsaturated fatty acids in the lipidsfacilitated the skin permeation of bioactive due to change in fluidity ofstratum corneum lipid structure caused by the packing nature ofunsaturated fatty acids [20,29]. Moreover, hepatitis B surface antigen

21 Days after last booster

IgG1 IgG2a IgG

b1 ND b1b 2.62±0.59bc 1.83±0.38cd 4.80±0.25bc

1.67±0.04 1.76±0.28 2.98±0.102.78±0.43 1.38±0.46 3.41±0.564.42±0.32a 2.4±0.56b 4.46±0.34b

lb/c mice received HBsAg at day 0 and 3 consecutive days as priming and booster doses(0.025 mg) by subcutaneous route was also administered which served as controls.

according to Tukey's multiple comparison test. b(pb0.001), c(pb0.01) or d(pb0.02),'s multiple comparison test. Non responder mice were excluded.

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is 22 nm particle composed of protein, phospholipids, cholesterol andits esters; solubilization by surfactants present in niosomesmight alsohave played some role in enhancement of permeation of these particlethrough stratum corneum.

4. Conclusion

Topical immunization, i.e. non-invasive vaccination enabling the useof a variety of antigens and adjuvant onto the skin (NIVS), provides arobust and novel approach of vaccination. Topical immunization usingniosomes based delivery system with application of the nontoxic cell-binding B subunit (CTB) of cholera toxin as an adjuvant is simple, stable,and potentially safe for topical immunization. It would increase the rateof vaccine compliance and greatly facilitate the successful implemen-tation of worldwide mass vaccination campaign against infectiousdiseases.

Acknowledgements

Authors are thankful to Shantha Biotechnics Ltd. (Hyderabad, India)for providing gift samples of recombinant HBsAg. Authors are alsothankful to All India Institute of Medical Sciences (AIIMS, New Delhi,India) for providing electron microscopy facility.

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