The most challenging task in the development of oral vaccine is to deal with physical and chemical instabilities of proteins. Protein instability is one of the major reasons why most of the vaccine are administered traditionally through injection rather than taken orally like most small chemical drugs (Wang, 1999). Moreover, problems, such as acid catalyzed degradation in the stomach, proteolytic breakdown in the gastrointestinal (GI) tract, poor perme-ability across the GI mucosa, and first-pass metabolism during transfer across the absorption barrier and in the liver must be overcome for the efficient delivery of anti-gen to immune cells (Liu et al., 2003). In order to achieve the successful oral delivery of proteins, they need to be protected from the harsh environment in the stomach. For designing vaccine, the formulator must consider that the natural pH environment of GI tract varies from acidic (pH ∼1.2) in the stomach to slightly alkaline in the
intestine (pH ∼7.4) (Shargel & Yu, 1999). Various authors have reported successful use of chitosan nanoparticles (CNPs) for mucosal vaccine delivery (Goyal et al., 2008). It can not only avoid deleterious preparation conditions but can also provide easy antigen loading onto the parti-cle surface (Hejazi & Amiji, 2002). Moreover, its superior properties including nontoxic, biodegradable, mucoad-hesive, and relatively mild formulation conditions make it an excellent polymer for antigen delivery via oral route. However, chitosan can lose its properties due to proto-nation in acidic conditions leading to burst release and deterioration of protein in gastric conditions (Schnurch et al., 1998; Vila et al., 2002; Lubben et al., 2003).
To avoid this problem antigen-loaded CNPs were coated by an acid-resistant polymer sodium alginate. This is polyanionic in nature and, therefore, bears oppo-site charges to chitosan. Therefore, mere incubation is sufficient for efficient coating on CNPs. Moreover, it is
ReseaRch aRtIcle
Microfold-cell targeted surface engineered polymeric nanoparticles for oral immunization
Basant Malik1, Amit K. Goyal1, T.S. Markandeywar1, Goutam Rath1, Foziyah Zakir1, Suresh P. Vyas2
1Nanomedicine Research Centre, Department of Pharmaceutics, Indo-Soviet Friendship College of Pharmacy, Moga, Punjab, India, and 2Department of Pharmaceutical Sciences, Dr. H. S. Gour University, Sagar (M.P.), India
abstractPresent work was envisaged to develop novel M-cell targeted polymeric particles that are capable of protecting the antigen from harsh gastric conditions. Ulex europaeus agglutinin (UEA-1) lectin was anchored for selective delivery of antigen to gut-associated lymphoid tissue (GALT). In the present investigation, chitosan nanoparticles were prepared by ionic gelation followed by antigen (bovine serum albumin, BSA) adsorption. Developed nanoparticles were further coated by UEA-1 lectin conjugated alginate and characterized for size, shape, zeta-potential, entrapment efficiency, and in vitro release. The immunological response of the developed system were performed in Balb/c mice and compared with aluminium hydroxide gel-based conventional vaccine. Results indicated that immunization with UEA-1 lectin conjugated alginate-coated particles induces efficient systemic as well as mucosal immune responses against BSA compared to other formulations. Aluminium-based vaccine dominated throughout the study, while failed in case of mucosal antibody. Additionally, IgG1 and IgG2a isotypes were determined to confirm the TH1/TH2 mixed immune response. The developed formulation exhibited superior systemic response along with dominating mucosal immunity. These data demonstrate the potential of UEA-alginate-coated nanoparticles as effective delivery system via oral route.Keywords: Oral immunization, chitosan nanoparticles, alginate, M-cell targeting, vaccines
Address for Correspondence: Prof. S. P. Vyas, Department of Pharmaceutical Sciences, Dr. H. S. Gour University, Sagar (M.P.) 470003, India. Tel: +91 7582265525. Fax: +91 75822265525. E-mail: [email protected]; [email protected]
(Received 22 March 2011; revised 27 June 2011; accepted 04 October 2011)
biocompatible and biodegradable by nature and shows a good safety profile. Alginate showed dual release kinetics at acidic and alkaline GI pH. Release of macro-molecules from alginate coating at low pH solutions was significantly reduced which is precious in the develop-ment of an effective oral delivery system (Yotsuyanagi et al., 1987; Kim & Lee, 1992; Sugawara et al., 1994). Theoretically, alginate shrinks at low pH (gastric envi-ronment) resulting in restricted antigen release (Chen et al., 2004; Dobakhti et al., 2006). In gastric fluid, the hydrated sodium alginate is converted into a porous, insoluble so-called alginic acid skin. Once passed into the higher pH of intestine, the alginic acid skin is con-verted to a soluble viscous layer (George & Abraham, 2006). This pH-dependent behavior of alginate was utilized to control release profiles. However, the rapid dissolution of alginate matrices in the higher pH ranges may result in burst release of antigen and subsequently possible denaturation of the protein by proteolytic enzymes. Therefore, immediate delivery (targeting) of this effective system to immune cells will be an effective approach.
In the present study, we deliberated a very effective delivery system for oral vaccination. We utilized two nat-ural polymers, chitosan and sodium alginate for safe and effective antigen delivery. Furthermore, it was directed to specific immune cell in gut-associated lymphoid tis-sue (GALT) by Ulex europaeus agglutinin (UEA-1) lectin
anchored at periphery of the system. Selected UEA-1 lec-tin specifically targets to α-L-fucose moiety that is local-ized at the apical pole of Microfold cells (M-cell) (Clark et al., 1993; Gulberg et al., 2006; Malik et al., 2010). In our previous study, we used UEA-1 lectin conjugated to poly(lactic-co-glycolic acid) (PLGA) for M-cell targeted delivery. Efficient delivery of antigen to the mucosal immune induction site (M-cell of the mice) was reflected by the triggered immune response obtained with targeted nanoparticles. The M-cell-targeted vaccine-loaded nano-particles elicited significantly higher immune response compared to nontargeted nanoparticles (Gupta et al., 2007; Mishra et al., 2011).
In the present study, we explored the targeting potential of UEA-1 with chitosan and alginate. The UEA-1 is covalently bonded on the surface of alginate-coated CNPs (ACNPs). Alginate was chemically modi-fied utilizing well-established aqueous carbodiimide chemistry (Figure 1) (Montalbetti & Falque, 2005). The 1-ethyl-3-[3 dimethylaminopropyl] carbodiim-ide (EDC) was used to form amide linkages between amino group of UEA-1 and the carboxylate moieties on the alginate backbone. The co-reactant N hydroxysul-fosuccinimide (NHS) was added to stabilize the reac-tive EDC-intermediate against a competing hydrolysis reaction, raising the efficiency of amide bond forma-tion. Bovine serum albumin (BSA) was used as model antigen throughout the study.
Figure 1. Scheme depicting the modification of alginate to anchor UEA-1 lectin.
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Materials and methods
MaterialsChitosan (medium molecular weight, 75%–85% deacety-lated), sodium tripolyphosphate (TPP) (85%), sodium alg-inate, UEA-1 lectin, BSA, NHS, and EDC were purchased from Sigma-Aldrich Pvt. Ltd., USA. The BCA protein estimation kit was purchased from GENEI (Bangalore, India). All other chemicals were of analytical grade.
Preparation of CNPsThe CNPs were prepared by the ionic gelation method using anionic tripolyphosphate (TPP). Briefly, 0.2% w/v chitosan was prepared in 1% (v/v) acetic acid aqueous solution. Then, TPP was dissolved in distilled water at the concentration of 1mg/mL. Subsequently, TPP was added drop by drop into chitosan solution with con-tinuous stirring at 1000 rpm on magnetic stirrer. About 30 min later, nanoparticles were centrifuged (Sigma Labororzentrifugen 3K30, Germany) at 55,201 RCF for 50 min. Then, the supernatant was discarded and the sediment was redispersed in distilled water and finally the product was freeze dried for further use.
Loading of the particles with antigenFirst, a suspension of the freeze-dried CNPs in a phos-phate buffer (pH 7.4) was disaggregated uniformly in an ultrasound bath. Then loading was done by incubating the antigen (2mg/mL) with CNPs (10 mg/mL) under mild agitation at room temperature. The loading efficacy of the CNPs was calculated indirectly by quantifying the free protein by BCA-protein estimation kit (GENEI™, Bangalore, India) using standard protocol.
Preparation of alginate-coated CNPsAlginate coating was performed by the method reported by Borges and co-workers with slight modifications (Borges et al., 2005). Developed antigen-loaded CNPs were slowly added to sodium alginate solution under mild agitation for 20 min at room temperature. The sus-pension was then centrifuged for 20 min at 2208 RCF and the supernatant was discarded.
Modification of alginate by grafting the targeting ligandsAlginate was chemically modified utilizing aqueous car-bodiimide chemistry as described by Montalbetti and Falque (2005). Carbodiimide chemistry was performed in 1% (w/v) alginate solutions in 0.1 M MES buffer at fixed pH (6.5) and NaCl concentrations (0.3 M) for 12 or 20 h. Sulfo-NHS was dissolved in the alginate solution at a ratio of 1:2 to EDC. Finally lectin was added after 5 min stirring and the reaction was carried out for 20 h. The alginate prod-uct was purified by dialysis against double distilled water for 4 days. Finally, alginate conjugated UEA-1 lectin was evaluated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using SDS electrophore-sis assembly (Bio-Rad, USA). After migration, the gel was
removed and stained with silver stain to locate respective position of proteins bands.
Characterization of developed formulations
MorphologyThe morphology and surface appearance of the particles were examined by scanning electron microscopy (SEM). The completely dried samples were coated with a thin gold layer and observed with SEM (Jeol JSM- 6100, JEOL BV, Schiphol-Rijk, Netherlands).
Size and zeta potential measurementsThe mean diameter and zeta potential of the nanoparti-cles were determined by Zetasizer Nano ZS 90 apparatus (Malvern Instruments, UK). The zeta potential analysis was performed using samples diluted with 1 mM HEPES buffer (adjusted to pH 7.4 with 1MHCl) in order to main-tain a constant ionic strength. For each sample the mean values ± SD of three determinations were established (Jain et al., 2009).
Protein loading efficiencyThe loading efficiency of the developed formulation was detected by quantifying the free BSA remained in the supernatant. Suspension was centrifuged (Sigma Labororzentrifugen 3K30, Germany) at 2208 RCF for 20 min and the amount of BSA in the supernatant was measured by BCA™ kit. The supernatant of blank formula-tion was adopted as the blank to correct the absorbance value of the antigen-loaded formulation. The corrected optical density (OD) value was used to calculate the con-centration of BSA in the supernatant.
In- vitro release studiesThe antigen release rates from different formulations were determined at both simulated gastric fluid (SGF, pH 1.2) and simulated intestinal fluid (SIF, pH 7.4). To perform the in vitro release study, the individual vials containing the developed formulations and 5 mL of release medium were incubated at 37°C on a constant shaking mixer. Samples were withdrawn at appropriate time intervals and centrifuged at 55,201 RCF for 30 min. The amount of free antigen in the supernatant was quantified using BCA™ kit. Results were expressed as a percentage of total antigens loaded in the final formulation. Supernatant of blank formulation was used as control for the correction of the OD value of the samples analyzed with BCA protein assay. In vitro release experiments were repeated thrice.
Assessment of structural integrity of antigenThe SDS-PAGE was used to assess in-process stability of antigen. Results were compared with the native antigen and reference markers. The antigens released after 48 h from different formulations were used in the study. The samples were loaded onto SDS electrophoresis assembly (Bio-Rad, USA) using 5% stacking gel and 12.5% separa-tion gel, run at 60–110 V until the dye band reached the
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M-cell targeted polymeric particles for oral immunization 79
gel bottom. After migration, the gel was removed and stained with Coomassie blue to locate respective posi-tion of proteins, which was then destained.
In vivo studiesThe immunogenicity of all the formulations was assessed using Balb/c mice (6–8 weeks of age). Animals were housed in groups of six (n = 6) with free access to food and water. They were withdrawn of any food intake 3 h before immunization. The study protocol, as approved by Institutional Animals Ethical Committee of ISF College of Pharmacy, Moga was followed. The studies were car-ried out as per the guidelines of Council for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Social Justice and Empowerment, Government of India. The animals were immunized according to the following protocol (Table 1). Each group received a booster dose after 2 weeks of primary immunization.
Sample collectionBlood was collected by retro-orbital puncture (under mild ether anesthesia) after 2, 4, and 6 weeks of primary and secondary immunization and serum was stored at −40°C until tested by enzyme-linked immunosor-bent assay (ELISA) for antibody. The nasal, vaginal, and salivary secretions were collected after 6 weeks of immunization. A vaginal wash was obtained following the method. Briefly, 50 μL of PBS containing 1% (w/v) BSA (1% BSA–PBS) was introduced into the vaginal tract of non-anesthetized mice using a Gilson pipette. These 50 μL aliquots were withdrawn and reintroduced nine times. A second vaginal wash was collected the follow-ing day and pooled with the first one. The nasal wash was similarly collected by cannulation of the trachea of sacrificed mice. The nasal cavity was then flushed three times with 0.5 mL of 1% BSA/PBS (pH 7.4). Salivation was induced as described in our previous studies (Jain et al., 2005). Mice were injected with 0.2 mL sterile pilocarpine solution (10 mg/mL) intraperitoneally. The saliva from mice after 20 min was collected using capillary tube. These fluids were stored with 100mM phenylmethyl sulfonyl fluoride (PMSF) as a protease inhibitor at −40°C until tested by ELISA for sIgA titre.
Measurement of specific IgG and IgA responseThe anti-BSA antibodies in blood samples were deter-mined by ELISA kit (Sigma-Aldrich Pvt. Ltd., USA). The IgG1 and IgG2a isotypes and IgA titre were determined
at day 42 using sigma kit (Sigma-Aldrich Pvt. Ltd., USA). Antibody response was plotted as log anti-BSA titre ver-sus time in days. Secretory IgA level in mucosal fluids was determined by ELISA. The end point titre was expressed as the reciprocal of the last dilution, which gave an OD at 490 nm above the OD of negative control.
Confocal microscopyConfocal microscopy was performed to confirm local-ization of the selected carrier constructs in the GALT. Fluorescein isothiocyanate-BSA (FITC-BSA) was used as fluorescent marker. The FITC-BSA-loaded formula-tions were administered orally to fully awake mice. The mice were administered with different formulations suspended in 250 μL of water. After 4 h of the administra-tion of dose, mice were killed by cervical dislocation and intestinal payer’s patch were removed, cut into pieces and washed with Ringer’s solution. The tissue were blot-ted and wiped with tissue paper. The tissues were fixed in Carnoy’s fluid (Absolute alcohol: Chloroform: Acetic acid, 6:3:1 v/v). The tissue blocks were prepared with paraffin wax, which were subjected to microtomy and mounted on slides and were analyzed under confocal microscope (Nikon, Japan).
Statistical analysisAll data were expressed as mean ± standard deviation. Significance was tested using Dunnett compare all ver-sus control. A p-value less than 0.05 was considered to indicate statistical significance for all comparisons.
Results and discussion
Characterization of modified alginateThe UAE-1 lectin conjugated alginate was synthesized by aqueous carbodiimide chemistry. The EDC was used for amide bond formation. The carbodiimide reacts with the carboxylic acid to form the O-acylisourea mixed anhy-dride. This intermediate can then directly react with the amine to yield the desired amide. Further, co-reactant N-hydroxysulfosuccinimide (sulfo-NHS) was added during carbodiimide reaction that stabilizes the reac-tive EDC-intermediate against a competing hydrolysis reaction, raising the efficiency of amide bond forma-tion. Schematic presentation of the synthesis is shown in Figure 1.
The SDS-PAGE was used to evaluate the conjugation of UEA-1 lectin with sodium alginate. Results were com-pared with the native lectin. The PAGE was run with the
wells containing standard markers (lane 1), native UEA-1 lectin (lane 2), and UEA-1 from lectin alginate conjugated (lane 3) Figure 2. Presence of extra bands with higher molecular weight in case of modified lectin compared with pure lectin indicating the successful conjugation of the lectin on alginate backbone.
Characterization of developed formulations
Size, shape, zeta potential, and entrapment efficiencyThe CNPs were prepared by ionic gelation method using TPP as cross-linking agent that has been described by several authors (Cremer & Kreuter, 1996; Wang et al., 2005). Ionic gelation is most widely used process for the formulation of CNP. The electrostatic interac-tion between amine group of chitosan and negatively charged group of polyanion such as tripolyphosphate leads to formation of CNPs (Tiyaboonchai, 2003). Further developed CNPs were loaded with the antigen. Electrostatic interaction between negative-charged antigen and positive-charged chitosan was clearly demonstrated by increase in particle size from 257.5 nm to 278.2 nm and decrease in zeta potential from 45.2 mV to 36.8 mV (Table 2). This may be attributed to the complete ionization of carboxyl group of antigen and amino group of chitosan at pH 7.4 (Phosphate buffer). We achieved sufficiently high loading efficiency (> 90%) by mere incubation for 30 min.
Antigen-loaded chitosan formulation was further coated with alginate that could provide better protec-tion in GIT environment and could enhance site-specific uptake via GALT. Similarly, adsorption of alginate onto the surface of chitosan could also be explained by electro-static interaction between the carboxyl group on alginate with the amino groups of chitosan resulted in forming strong interactions (Borges et al., 2005). Zeta potential of both alginate-coated chitosan (−24 ± 3.22 mV) nanoparti-cles as well as modified alginate-coated (−28.6 ± 4.51 mV)
compared to plain chitosan (+45.2 ± 3.04mV) has clearly demonstrated strong interaction and adsorption of alg-inate over the surface of chitosan. However, our studies have shown antigen desorption (not significant) during the adsorption of alginate over antigen-coated chito-san nanoparticles. This may be due to the competitive adsorption of alginate and antigen on chitosan surface.
Finally, optimized formulations were evaluated for size, zeta potential in triplicate and compared. The mean par-ticle size of optimized CNP was about 257 nm and the PDI was 0.241 (Table 2). The SEM studies were conducted which clearly indicate spherical shape of ACNPs (Figure 3).
In vitro protein releaseAny orally administered formulation encounters both simulated gastric and simulated intestinal pH. The sur-face of CNPs was suitably modified to prevent acid degra-dation of antigen in stomach and to controlled release of antigen in the intestine. To rationalize the alginate coat-ing, release studies were carried out at pH 1.2 (SGF, Figure 4) and pH 7.4 (SIF, Figure 5) pH of antigen from conven-tional CNPs as well as alginate-coated nanoparticles. In contrast to uncoated CNP, the release profile of antigen from nanoparticles was significantly modified after coat-ing with sodium alginate, although at pH 7.4 there was no significant difference (≈ 10%) in cumulative percent-age antigen release from both the formulations after 12 h. This slight difference might be due to direct exposure of antigen to release medium in case of uncoated CNP which could be easily desorbed at ionic environment. The difference was highly significant at pH 1.2 initial burst release (about 80%) of antigen from uncoated CNPs within the first 10 min, whereas the difference was only 30% from alginate-coated CNP after 6 h. Repulsive forces due to protonation of both chitosan and BSA at acidic pH may be responsible for burst release. However alginate coating might be responsible for controlled release of the antigen from coated formulation. The results indicate
Figure 2. SDS-PAGE analysis of standard marker, pure lectin and lectin from conjugated alginate. Figure 3. SEM photograph of ACNP (4000×).
that alginate coating provides sufficient protection to antigen in gastric environment.
Structural integrity of antigenIn the process, stability and integrity of the entrapped antigen was evaluated using SDS-PAGE. Since process conditions may affect the three-dimensional structure of protein, the BSA released from the formulations were compared with pure BSA for any change in structural integrity. The PAGE was run with the wells containing standard markers (lane 1), native BSA (lane 2), antigen released from CNP (lane 3), alginate-coated CNP (lane 4), and lectin conjugated alginate coated (lane 5) after 48 h. Visible bands for pure as well as released antigens from the formulations were identical (Figure 6) and confer favor-able processing conditions. This reveals that the prepara-tion conditions do not cause any irreversible aggregation or cleavage of the protein. For the comparison, the native
BSA and molecular weight marker were also run in sepa-rate wells of the same gel.
Immunological studiesTo evaluate the immune adjuvant capability of the developed formulations, immunization studies were performed. Different groups were designed for the immu-nological study each containing six mice. Group I kept as controlled, Group II received conventional CNP-based vaccine, and Group III–IV received alginate-coated for-mulations via oral route. Group V received aluminium-based vaccine via parenteral route. Each group received a booster dose after 2 weeks of primary immunization. Blood samples were collected by retro-orbital plexus and IgG titre was estimated by ELISA. The end point IgG titre was expressed as the reciprocal of the last dilution, which gave an OD at 490 nm above the OD of negative con-trol. Figure 7 shows the IgG titre obtained with different
Figure 4. Antigen release in simulated gastric fluid (SGF).
Figure 5. Antigen release in simulated intestinal fluid (SIF).
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formulations at different time interval. Immunological outcomes clearly indicate significantly improved (p < 0.001) immune response of mice immunized orally with alginate-coated formulations (Figure 7).
Among all the orally administered formulations, lectin-anchored ACNPs (LACNP) exhibited more sig-nificant results (IgG titre). This could be attributed to collective protective effect of alginate coat barrier against physicochemical biochallenges and targeting of UEA-1 lectin to α-L-fucose moiety expressed on apical membrane of M-cell that might have resulted in better presentation of antigen to mucosal immune system. However, plain CNP-based formulation gives poor immunological response. Low IgG titre with CNPs can be attributed due to the susceptibility of chitosan to gastric acid resulting in dissolution of polymer and subsequent exposure of antigen to acidic pH and prote-olytic enzymes. Moreover, aluminium-based formula-tion dominated in the IgG titre throughout the studies and LACNP responses were found to be only com-parative to them. Interestingly parenteral aluminium-based vaccine failed in mucosal response (sIgA), while LACNP-based formulation dominated in this counter-part also (Figure 8). It may be attributed to the fact that parenterally administered antigen lacks the ability to stimulate mucosal immune system.
Figure 6. SDS-PAGE analysis of standard marker, pure BSA and BSA from formulations.
Figure 7. IgG antibody levels in serum on immunization with different formulations.
Figure 8. IgA antibody levels in serum on immunization with different formulations.
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M-cell targeted polymeric particles for oral immunization 83
Figure 8 depicts secretory IgA levels in salivary, nasal, and vaginal secretions. It can be inferred from the data that lectin-based formulation efficiently elicited mucosal immune response (sIgA level) than nontargeted formula-tions. Negligible IgA response was observed on intramus-cular administration of aluminium-adsorbed vaccine. It may be attributed to the fact that parenterally administered antigen lacks the ability to evoke B cell isotype switch-ing responsible for mucosal immune response. Thus, the orally administered system can provide additional advan-tage of mucosal immunity, i.e. neutralizing the antigen at first exposure. Mucosal IgA plays a significant role in pro-tection against enteropathogens and viruses (McKenzie et al., 2004). Secretory IgA (sIgA) inhibit antigen bacterial adhesion, neutralizes viruses, bacterial toxins, and thus imparts nonspecific defense mechanism. Antigens are eliminated from the tissue through binding to IgA and subsequent polyimmunoglobulin receptor-mediated transport of immune complexes through epithelial cells (Holmgren & Czerkinsky, 2005). Specific sIgA is vital in providing the protection against mucosally invading bac-teria and viruses.
To generate a complete immune response, the system should be able to produce the humoral as well as cellular immunity. The generation of dominant TH1/TH2-based immunity is important for eradication of any mucosal infection (Neutra et al.,1996; Constant & Bottomly,1997; Banchereau et al., 2000). In the present study, the IgG1 and IgG2a isotypes were determined at day 42 to assess
the TH1/TH2 immunity pattern (Figure 9). Aluminium-based formulation gave high level of IgG1 isotype indi-cating good TH2 or humoral immunity, whereas failed to generate considerable IgG2a level i.e. hallmark of TH1 immunity. Alginate-coated formulation gave mixed TH1/TH2 response with good IgG2a/IgG1 ratio. However lectin-based formulation was able to generate highly sig-nificant cell mediated immunity when compared to plain CNP and aluminium adsorbed parenteral vaccine. Thus developed lectin-alginate based systems have shown capability enough to target mucosal immune induction site, so as to generate both arm of immune response after mucosal administration.
Peyer’s patch uptake studyConfocal fluorescence results revealed quantitative targeting of nanocarrier constructs. It was noted that intensity of fluorescence in case of targeted formulation (LACNP) was high compared to plain alginate-coated nanoparticles as well as uncoated CNPs. This may be attributed to the specificity of UEA-1 lectin-based for-mulation toward M-cells that imparted specific localiza-tion of the nanoparticles. On the other hand, in case of uncoated nanoparticles, fluorescent marker was released in the vicinity of the M-cell due to mucoadhesive property of chitosan. This leads to release of entrapped fluorescent marker in the wide delocalized space as is evident from the photomicrographs which is highly undesirable for successful vaccination (Figure 10).
Figure 9. IgG1 and IgG2a anti-BSA antibody isotopes at day 42 in sera of mice.
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conclusion
The ACNP have been frequently used as potential carrier system for oral vaccination. Similarly, UEA-1 has been found to be a specific ligand for α-L-fucose present at the apical surface of M-cells. Furthermore, exploring their combination has proven worthwhile by our stud-ies conducted. The UEA-1 anchored ACNPs resulted in the elicitation of a strong systemic as well as mucosal immune response followed by ACNP. Whereas, CNP were least effective probably because of their suscepti-bility to harsh GI challenges. Therefore, UEA-1 lectin-based ACNPs seems to be a promising technology for needle-free targeted vaccination, which can be utilized for safe and effective vaccine delivery via oral route. The approach is not antigen specific, while equally appli-cable to each antigen, with slight modification during antigen loading.
acknowledgment
Authors are thankful to Prof. S. C. Lakhotia and Prof. J. K. Roy (Banaras Hindu University, Varanasi, India) for Confocal Laser Scanning Microscopy and Sophisticated Analytical Instrumentation Facility (Punjab University, Chandigarh, India) for electron microscopy.
Declaration of interest
Author Basank Malik, acknowledges Department of Biotechnology (DBT), New Delhi for financial assistance in the form of Senior Research Fellowship.
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