Development and characterization of alginate coated low molecular weight chitosan nanoparticles as new carriers for oral vaccine delivery in mice

Accepted Manuscript Title: Development and characterisation of alginate coated low molecular weight chitosan nanoparticles as new carriers for oral vaccine delivery in mice Author: Subrata Biswas Mainak Chattopadhyay Kalyan Kumar Sen Malay Kumar Saha PII: DOI: Reference:

S0144-8617(14)01240-5 http://dx.doi.org/doi:10.1016/j.carbpol.2014.12.044 CARP 9547

To appear in: Received date: Revised date: Accepted date:

31-1-2014 9-12-2014 10-12-2014

Please cite this article as: Biswas, S., Chattopadhyay, M., Sen, K. K., and Saha, M. K.,Development and characterisation of alginate coated low molecular weight chitosan nanoparticles as new carriers for oral vaccine delivery in mice, Carbohydrate Polymers (2015), http://dx.doi.org/10.1016/j.carbpol.2014.12.044 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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 Alginate coating onto CS LMW nanoparticles protect antigen in acidic

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106 kDa).

condition.

 Degree of toxicity was not affected significantly due to difference of MW of

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 Nanoparticles were prepared by using different low MW of CS (42, 74 and

CS.

 Significant correlation was observed between the immune response with CS

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Highlights

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Development and characterisation of alginate coated low molecular weight chitosan nanoparticles as new carriers for oral vaccine delivery in mice

15 Subrata Biswas a, Mainak Chattopadhyay b, Kalyan Kumar Sen c,*, Malay Kumar

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Saha a

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a

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Research, Kolkata– 700010, India b Department of Pharmacology, Bengal School of Technology, Hooghly-712102, West

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National Institute of Cholera and Enteric Diseases, Indian Council of Medical

Bengal, India.

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c

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More, G.T. Road, Asansol-713301, West Bengal, India.

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Department of Pharmaceutics, Gupta College of Technological Sciences, Ashram

25 26 Email addresses:

SB- [email protected]

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MC- [email protected]

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KKS- [email protected]

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MKS- [email protected]

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*Corresponding author

Dr. Kalyan Kumar Sen Principal & Professor Gupta College of Technological Sciences Ashram More, G T Road, Asansol - 713301 West Bengal, India E-mail: [email protected] Telephone No.: +91-341- 2312130 Fax No.: +91-341- 2314604

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ABSTRACT

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In the present study, nanoparticles of low MW chitosan (CS) were formulated in

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which measles antigen was entrapped and subsequently coated with sodium

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alginate. The size and surface properties of the nanoparticle can be tuned with

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different MW of CS. In vitro release studies showed initial burst release followed by

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extended release, best fitted in the Makoid-Banakar model (R2 > 0.98). SDS-PAGE

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assay revealed that alginate coating could effectively protect antigen in acidic

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condition for at least 2 h. Cell viability was assessed using MTT assay into HT 29 cell

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line. Formulations were orally administered to mice and immunological responses

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were evaluated using ELISA method. Obtained results showed that measles antigen-

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loaded CS nanoparticles induced strong immune response and significant correlation

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was observed between the immune response with CS MW. Protecting ability of

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antigen in gastric environment, sustained release kinetics, systemic and mucosal

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immune responses and low cytotoxicity observed for the alginate coated

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nanoparticles demonstrated that LMW CS could be promising platform for oral

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vaccine delivery.

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Keywords: adjuvant, cytotoxicity, mucosal immunity, vaccine

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Abbreviations: PAGE- polyacrylamide gel electrophoresis, GALT-gut associated

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lymphoid tissue

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1.

Introduction

84 It is well established that protection against pathogenic organisms occurs better with

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the presence of mucosal antibody than with serum antibody (Baumann, 2008).

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Compared to traditional routes of administration, oral vaccine delivery systems offer

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several attractive advantages such as lower cost, ease of administration, higher

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patient compliance, reducing the need for trained personnel, averting vaccine-related

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infections correlated to the disposal and reuse of needles in systemic delivery as well

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as higher capacity of much for mass immunizations (Fooks, 2004; Kim, Lee, & Jang,

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2012; Kostrzak et al. 2009; Zhu, & Berzofsky, 2013). It has also been demonstrated

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that orally administered antigens can induce both local and systemic immune

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responses, providing a complete immune response (Neutra, & Kozlowski, 2006).

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However, immune responses following oral administration of vaccines are usually

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poor due to rapid degradation of antigen in the harsh gut environment and their poor

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uptake by appropriate target sites, namely M cells located in the Peyer’s patches

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(Czerkinsky, & Holmgren, 2009). Keeping this in mind, several studies have been

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performed to show that by associating the vaccine with a number of particulate

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delivery systems, the degradation of the antigen in the harsh environment of the GI

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tract is prevented and the uptake by M-cells is enhanced by several times (Delie, &

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Blanco-Prieto, 2005; Eyles, Sharp, Williamson, Spiers, & Alpar, 1998; van der

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Merwe, Verhoef, Verheijden, Kotze, & Junginger, 2004). In addition, biodegradable

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particles allow a sustained release of the antigen, increasing the duration of the

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contact between antigen and immune cells thus favouring an effective immune

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response.

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In the past decades, chitosan (CS) has been extensively used as a carrier

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candidate because of its excellent stability, mucoadhesive property, enhanced

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penetration capacity across the mucosal barriers and good compatibility with vaccine

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antigen (Borges et al., 2007; Lee et al., 2011; Yoo et al., 2010; Zaharoff, Rogers,

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Hance, Schlom, & Greiner, 2007). The formation of CS nanoparticles is a process

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based on the complexation of oppositely charged molecules by electrostatic

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interactions (Csaba, Koping-Hoggard, & Alonso, 1997). Nanoparticles can be easily

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constructed using an ionic gelation process that involves sodium tripolyphosphate as

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a negatively charged molecule and CS as polycation (Calvo, Remunan-Lopez, Vila4 Page 4 of 32

Jato, & Alonso, 1997; Oliveria et al, 2012). The mild technique involves the mixing of

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two aqueous solutions at ambient temperature while stirring without using sonication

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or organic solvents. Proteins with low isoelectric point, such as BSA and measles

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antigen, were better associated with the nanoparticles when dissolved in the alkaline

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sodium TPP solution (Li et al., 2008). Although CS nanoparticles have numerous

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advantages as delivery carriers for oral vaccination, their limited ability to control the

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release of encapsulated antigens and easy solubility in acidic medium largely

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hamper their development (Li et al., 2008). These obstacles may be overcome by

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coating those antigen loaded particles with an acid-resistant polymer, like sodium

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alginate (Borges et al., 2006).

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Sodium alginate, an anionic polysaccharide with favourable biological

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properties, can be easily interacted with cationic CS nanoparticles to form an

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electrolyte complex via electrostatic interactions (Du et al., 2013; Kim et al., 2002).

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Alginate-CS polyionic complexes form through ionic gelation via interactions

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between the cationic amine groups of CS and the anionic carboxyl groups of alginate

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(Gazori et al., 2009). This coating procedure was performed at relatively mild

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conditions without using any organic solvent at room temperature, enabling antigens

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to be incorporated into the CS/alginate matrices with retention of biological activity

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(Wee, & Gombotz, 1998). Calcium ion is used as crosslinking agent to strengthen

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and stabilize the particles (Borges et al., 2006). Recently, Liu et al. (2013)

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demonstrated that DNA loaded in alginate coated CS nanoparticles alginate-coated

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CS nanoparticles containing DNA are effectively taken up and expressed by the

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Payer’s patches. Release profiles of vaccines from these delivery systems showed

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that burst release of loaded BSA in pH 1.2 (simulated gastric fluid) was largely

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prevented by its entrapment in alginate-coated CS particles (Anal, Bhopatkar,

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Tokura, Tamura, & Stevens, 2003). Moreover, Borges et al. (2006) demonstrated

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that sodium-alginate coated nanoparticles were readily able to be taken up by rat

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Peyer’s patches which is one of the essential features to internalize, deliver and

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target the intact antigen to specialized immune cells from gut associated lymphoid

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tissue (GALT). In vivo studies have shown higher antibody titre for alginate coated

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CS nanoparticles compared to plain CS nanoparticles (Malik, Goyal, Zakir, & Vyas,

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2011). This delivery system also acted as an effective adjuvant for hepatitis B

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surface antigen when subcutaneously administered in a mouse model (Borges et al.,

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2008). It has been demonstrated that transfection efficiency is closely related to the

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MW of polymer. CS of 10 -150 kDa with a specific degree of deacetylation allows

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maximum transgenic expression in vitro and CS nanoparticles less than 500 nm in

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diameter are transported through an endocytotic mechanism (Godbey, Wu, & Mikos,

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1999; Lavertu, Methot, Tran-Khanh, & Buschmann, 2006; Liu et al., 2013). Recently, there has been growing interest in low molecular weight CS (CS-

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LMW) as a new mucosal delivery vehicle due to its greater solubility in neutral

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aqueous solvents with low viscosity and faster degradation than higher molecular

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weight CS counterparts (Amoozgar, Park, Lin, & Yeo, 2012; Fernandes et al., 2012;

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Liu et al., 2013). Vila et al. (2004) reported that nanoparticles made of CS-LMW with

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tetanus toxoid (TT) as a model protein could be produced by an ionic-cross linking

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technique and proving to be promising carrier for nasal vaccine delivery. Following

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intranasal administration, TT-loaded nanoparticles elicited an increasing and long-

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lasting humoral response as compared to the soluble antigen. Plasma or serum

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samples have traditionally been used for generation of serological data due to the

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difficulties associated with handling whole blood. However, this approach requires

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relatively large volumes (200 - 1000 µl) of blood in order to produce the required

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volume of matrix for bioanalysis, which makes it difficult to generate serial profiles in

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mice test models. Composite profiles are therefore necessary, which can result in

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lower quality data and requires the use of a greater number of animals. Conventional

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ELISA are formatted to be used only for determining whether a given sample

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contains antibody at a concentration above or below a set threshold value, although

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quantification of antibody response is prerequisite to monitor the progress after

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immunization. In recent years, dried blood spot (DBS) assays have received

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increasing attention as an alternative to venous blood sampling and can be adopted

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in preclinical serological studies.

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To the best of our knowledge, there is no published data whereby alginate

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coated CS-LMW nanoparticles are explored as vehicles for the oral administration of

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vaccine. In this study, we have tried to develop a novel oral vaccine carrier based on

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alginate coated CS-LMW nanoparticles to meet the requirement of oral vaccine. So

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the objectives of the studies are to develop the nanoparticle based oral vaccine

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delivery system and the delivery system will be adopted to evaluate the

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physicochemical properties, loading efficiency of the particles and the stability of

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antigen loaded nanoparticles against acidic condition. Cytotoxicity will be evaluated

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using HT 29 human epithelial cell lines for the different formulation of nanoparticles. 6 Page 6 of 32

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Finally, the effects of molecular weight of CS (42, 74 and 106 kDa) on the ability of

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these nanoparticles will be studied in a mouse model to elicit significant immune

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response as measured using DBS ELISA method.

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2.

Experimental

2.1.

Materials

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CS-LMW of three different molecular weight (42, 74 and 106 kDa) were

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obtained through oxidation-reduction reaction of high molecular weight CS (MW 375

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kDa) (Sigma Aldrich, USA) using NaNO2 as described by Mao et al. (2004). Briefly,

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varying quantities of 0.1 M NaNO2 were added to the CS solution with controlled

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temperature at 25 ºC under magnetic stirring, and the reaction was left overnight to

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assure completion of the degradation. The depolymerisation process of CS has been

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previously simulated based on Box-Benkhen design experiments. With this

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approach, the required amount of NaNO2 could be predicted in order to obtain

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approximate molecular weight of 42, 76 and 106 kDa. The molecular weight of the

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CS was determined using High Pressure Size Exclusion Chromatography (HPSEC).

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Sodium tripolyphosphate (TPP), sodium alginate, CaCl2, DMEM (Dulbecco’s

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Modified Eagle’s Medium), HEPES [N-(2-hydroxyethyl piperazine-N-(2-

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ethanosulphonic acid)], MTT (methylthiazolyl diphenyl-tetrazolium bromide)

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reagents, goat anti-mouse IgG peroxidise conjugate and TMB/H2O2 substrate for

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ELISA were procured from Sigma (USA). Anti measles monoclonal antibody was

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purchased from Millipore. BCA kit was purchased from Pierce (USA). Measles

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antigen was kindly donated by Serum Institute of India Ltd (Pune, India). 96-well flat

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bottom polystyrene plate was purchased from Nunc (Denmark). All other reagents

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were of analytical grade. Ultrapure water (MilliQ, Waters, USA) was used to prepare

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all solutions and freshly prepared solutions were used in all experiments.

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Human intestinal epithelial cells (HT 29) were cultured and maintained in

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DMEM with supplemented with 10% fetal bovine serum and 1% antibiotic-

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antimycotic solution (Invitrogen, Carlsbad, CA). Cells were maintained in 5% CO2 at

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37 ºC humidified incubator for 10 days.

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Immunogenicity of the antigen was evaluated in 6 weeks old specific

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pathogen-free male BALB/c mice. Animal care and handling were maintained

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according to the guidelines established by the Institutional Animal Ethical Committee 7 Page 7 of 32

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(IAEC) of Gupta College of Technological Sciences, India. The experiment protocols

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were approved by IAEC and the experimental procedures were in accordance with

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IAEC.

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Preparation of CS nanoparticles

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CS nanoparticles were prepared by an ionotropic gelation process technique

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using TPP as a crosslinking agent under stirring (Li et al., 2008). Briefly, 0.2% (w/v)

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CS solution was prepared in 25 mM sodium acetate buffer (pH 5.2) and TPP was

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dissolved in ultra-pure water in order to obtain 0.84 mg/ml solutions. Nanoparticles

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were formed when 10 ml TPP solution was added drop wise to a 10 ml CS solution

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and magnetically stirred at 300 rpm during 30 min. For association of measles

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antigen with CS nanoparticle, 600 µg of measles antigen was incorporated in the

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TPP solution. Cryoprotectant mannitol (1:1) was added to the resulting nanoparticle

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suspension, frozen at -70 ºC for 48 h, and then lyophilized in a freeze dryer (FDU-

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S603, Operon, Korea). Freeze-dried CS nanoparticles were redispersed in ultrapure

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water for further use.

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234 2.3.

Antigen loaded CS nanoparticle suspension (20 ml, pH 5.1) was added drop-

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Preparation of alginate coated CS nanoparticles

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wise to sodium alginate solution (20 ml, pH 7.0) at concentration of 1 mg/ml under

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mild stirring for 30 min at room temperature. Alginate coated CS nanoparticles were

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redispersed into aqueous solution of CaCl2 (pH 7.0, 0.5 mmol/l) to crosslink the

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alginate layer on the surface of antigen loaded CS nanoparticles. Cryoprotectant

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mannitol (1:1) was added to the resulting suspension and stored at -70 ºC for 48 h

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before freeze-drying.

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2.4.

Loading capacity of antigen in coated nanoparticles

Loading capacity (LC) of measles antigen loaded CS nanoparticles were was

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calculated in an indirect way by determining the measles antigen remaining in the

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aqueous phase using the BCA kit (Borges et al., 2006). Aliquots of the resulting

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nanoparticle suspension were centrifuged for 20 min at 18,000 X g and the

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supernatants were then separated from the nanoparticles. The supernatant of blank

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CS nanoparticles was adopted as the blank to correct the absorbance reading value

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of the measles antigen loaded nanoparticles. The LC values of antigen loaded

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nanoparticles were calculated from the following equations:

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2.5.

The morphological characteristics of nanoparticles were examined by

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Morphological characterization, size and surface charge

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scanning electron microscopy (JEOL JSM 5200, Japan). The particle size

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distribution was measured by laser diffraction and zeta potential of the particle was

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examined by zeta analyser using Zetasizer (Ver. 6.00, Malvern Instruments Ltd, UK)

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with ultrapure water as solvent (pH 7, 25 OC). These measurements were run at

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least three times (replicates) and were presented as mean ± SD with independent

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particle batches.

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2.6.

In vitro release studies

Approximately 20 mg dried nanoparticle samples (CS or alginate-coated CS )

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CS nanoparticles and alginate coated CS nanoparticles were incubated in 3 ml of

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phosphate buffered saline (PBS, pH 7.4) in an Eppendorf tube, and then placed in

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an air shaker at 100 rpm at 37 OC. At predetermined time intervals, the samples

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were centrifuged at 13,200 rpm for 20 min and the supernatant was replaced with

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fresh release medium to reach the original volume and resuspended. The amount of

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antigen released was determined by BCA reagents as described in section 2.4 2.3.

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According to protocol, the amount of antigen released was expressed as percentage

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of total antigen encapsulated in CS nanoparticles as calculated from the LC value. A

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sample consisting of only non-loaded nanoparticles re-suspended in PBS was used

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as a blank. In vitro release experiments were repeated three times.

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2.7. Stability study in simulated gastric fluid Different formulations of CS nanoparticles (antigen loaded CS nanoparticles

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and alginate coated antigen loaded nanoparticles) were incubated with 0.5 ml of HCl

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(0.01 M) in a shaker bath at 37 ºC for 2 h. Then, acid was neutralized by 0.5 ml of

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aqueous NaOH (0.01 M) solution. These mixtures were allowed to stand for another

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24 h, then diluted with PBS (pH 7.4) to a final volume of 4 ml. Twenty four hours

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later, supernatant containing released antigen was collected and analyzed by SDS-

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polyacrylamide gel electrophoresis (SDS-PAGE) in slab gel systems employed a

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7.5% running gel and a 5.0% stacking gel. The protein sample was prepared in

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electrophoresis loading-buffer (60 mM Tris-HCl, pH 6.8, with 25% glycerol and 2%

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SDS containing 0.1% Bromophenol blue solution, and h-mercaptoethanol 2-

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mercaptoethanol) and heated for 5 min at 95 ºC. The pure antigen was used as

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control. After electrophoresis, the protein bands were visualized by staining with

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Coomassie Blue.

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2.8.

In vitro cytotoxicity assay

To investigate the potential cytotoxicity of uncoated and alginate coated CS-

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antigen nanoparticles, the cell viability was analyzed using HT 29 cell lines in a MTT

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assay. The HT 29 human epithelial cell line was selected in the present study as a

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convenient in vitro model to assess polymer toxicity (Sergent, Paget, & Chevillard,

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2012). The assay was performed according to the method described by Mosmann

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(1983). HT 29 cells were seeded into 96-well microplates at a density of 3000

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cells/cm2. The medium was removed 24 h after plating and the cells were incubated

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for 6 h with 50 µl nanoparticle in HBSS (Nanoparticle concentrations were 0.25, 1.0

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and 4.0 mg/ml). SDS (10mg/ml) was used as positive control and HBSS as

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reference for 100% cell viability. After incubation for 6 h the medium was discarded,

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the cells were washed twice with phosphate-buffered saline and 50 µl of 5 mg/ml

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MTT solution in PBS were added to each well. The plates were incubated in a

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humidified atmosphere of 5% CO2 at 37 ºC for another 6 h. Subsequently, the wells

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were emptied, and the formazan crystals were dissolved by adding 100 µl dimethyl

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sulfoxide (DMSO) to each well. The absorbance was measured in triplicate at 570

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nm, with a background correction at 630 nm, using a microplate ELISA reader

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(Multiscan Ex, Thermo Scientific). The MTT assay was repeated three times.

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2.9.

Immunization studies The immunogenicity of the antigen was assessed in BALB/c mice following

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oral immunization. Mice were housed in groups of six (n = 6) animals for one week

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before the experiments were conducted. Mice were immunized orally with various

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formulations (Table 1) containing 20 µg of antigen (calculated from loading capacity 10 Page 10 of 32

of the nanoparticles) in 50 µl of saline solutions on days 0, 7, 14 and 28 via a 25-

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guage 1ml hypodermic needle (Song et al., 2005). Subcutaneous immunization was

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also carried out with measles vaccine to serve as positive control. One group of mice

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was treated with alginate coated blank bead beads to serve as negative control and

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measles antigen for another group was administered to another control group. All

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animals were conscious during the administration.

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2.10. Sampling of body fluids

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Blood samples were drawn from the tail-vein on day 7, 14, 28, 56 post

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administration using a lancet (Hoff, 2000). Three drops of blood were collected in the

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protein-savour dry blood spot (DBS) card (Biorad, USA). Blood spots were allowed

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to dry overnight at room temperature. To obtain intestinal lavage fluids, on day 28

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and 56 three mice were anesthetized i.p. with pentobarbital, then necropsied.

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Lengths of upper small intestine from each mouse were sectioned longitudinally, and

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the Intestinal lavage fluids were obtained by washing the intestine three times with

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0.5 ml of ice-cold saline containing the protease inhibitor. DBS card was kept in a

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zip-lock bag at room temperature with desiccant and gastric lavage was stored at -

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20 ºC until analyzed.

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2.11. ELISA

IgG antibody levels in each sample were determined using a DBS ELISA test.

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Discs of approximately 3 mm diameter were punched from DBS cards, individual

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paper discs were transferred to individual wells of a 96-well titre plates, and analytes

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were eluted with PBS containing 0.05% (w/v) Tween 20 (PBST) buffer. First,

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measles antigen (4 µg/ml) in carbonate buffer (pH 9.6) was added to microplates and

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incubated at 4 ºC in a humid container. The wells were washed three times with

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PBST. To minimize non-specific interactions, 100 µl of PBST containing 2.5% (w/v)

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of dried skimmed milk powder was added to the wells and incubated for 1 h at 37 ºC

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in a humid container and the plate were washed three times with PBST. Eluted

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samples were serially diluted 5-fold starting at either a 1:10 or 1:100 dilution and

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incubated 2 h with a known concentration of antigen, then were added to the wells

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and the plates were incubated for 2 h at 37 ºC in a humid container and washed. 11 Page 11 of 32

Then, 100 µl of anti-mouse IgG peroxidase conjugate, diluted 1:2000 in PBSTM

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buffer, was added into each well and plates were incubated for another 1 h at 37 ºC.

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The plates were washed, and 50 µl of substrate was added to each well, and the

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reaction was allowed to keep proceed in the dark for 15 min at RT room

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temperature. 100 µl of stopping solution (1 N sulphuric acid) was added to each well

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and plates were read at 450 nm on a microplate reader.

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Intestinal lavages were sonicated on ice to extract immunoglobulins from

mucin and centrifuged (16,000 X g, 60 min, 4 ºC). Supernatants were diluted with

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200 µl of PBS for ELISA assay of specific IgA content as described above for IgG.

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Titre was derived as the maximum dilution of intestinal lavages giving an OD of 0.2

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after correction for background. Reciprocal dilutions corresponding to endpoint titres

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were determined and were presented as mean log10titre ± SEM per group.

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2.12. Statistical analysis

If another method is not explicitly stated, the data were expressed as mean

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values ± standard deviation (SD). The effect of the CS molecular weight on the cell

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viability and adjuvaniticity adjuvant capacity was assessed with the aid of SPSS 16.0

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(SPSS Inc., Chicago, USA) using one way ANOVA with the significance level set at

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p < 0.05. Modeling and comparison of dissolution profile and release kinetics were

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evaluated using DDSolver.

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3.

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3.1.

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Results and discussion Formulation and characterization of CS LMW nanoparticles To achieve the nanoparticle formulation for measles antigen with CS LMW,

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systematic preformulation studies were carried out and the composition of this

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formulation was chosen as CS/TPP ratio of 2:0.84 and CS/sodium alginate ratio 2:1.

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Antigen loaded nanoparticles were systematically characterized to evaluate

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morphology, drug loading and releasing efficiency. The study of scanning electron

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microscopic images revealed that antigen-loaded alginate-coated CS nanoparticles

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have irregular shape with slight aggregation (Fig. 1). The particle sizes and zeta

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potentials of antigen loaded alginate uncoated and coated CS nanoparticles of three

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different MW are shown in Table 2. Decreasing the MW of CS resulted in a decrease

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in nanoparticle size and increasing loading efficiency (Lee, Lee, & Jon, 2010). 12 Page 12 of 32

However, very low MW CS may have poor loading efficiency and may not form

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stable nanoparticles (Fernandes et al., 2012). Vaccine carried with alginate coated

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CS nanoparticles exhibits similar trends in increasing particle size compared with CS

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nanoparticles in all three cases. The zeta potential of CS nanoparticles is an

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important factor for the stability and mucoadhesiveness of nanoparticles. Generally

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the higher zeta potential of particles led to more stability, whereas the lower zeta

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potentials of the particles induce agglomeration (Gan, Wang, Cochrane, &

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McCarron, 2005). CS molecular weight effect on encapsulation is largely the

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consequence of how the cationic CS chain interacts with protein molecules which

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have a more elaborate and complex 3-D structure as polypeptide chains contain

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both positive and negatively charged functional groups, and also hydrophobic

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regions which tend to fold inside the 3-D structure in an aqueous environment (Gan,

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Wang, Cochrane, & McCarron, 2005). However, encapsulation efficiency of a

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protein, tetanus toxoid, was high, irrespective of the CS molecular weight and

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indicated that the entanglement of the protein within the CS chains is not affected by

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their molecular weight within the range studied (Vila et al., 2004).

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Table 2

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Fig. 1.

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3.2

In vitro release studies

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The release profiles of antigen from uncoated and alginate coated CS

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nanoparticles in phosphate buffer are shown in Fig. 2. CS nanoparticle formulations

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exhibited two release phases, a rapid release over the first 3 h followed by a slow

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release for up to 120 h. The burst release might be attributed to the fact that antigen

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was loosely bound onto CS nanoparticles by ionic interactions which could be easily

411

desorbed in an ionic environment (Chen, Zhang, & Huang, 2007). The second phase

412

might correspond to those antigen molecules more efficiently entrapped and tightly

413

bound to the CS molecules. Antigen release was relatively rapid for the first 3 h in

414

case of lower MW of CS CS MW, which can be attributed to an easier detachment of

415

low MW CS molecules and antigen molecules located at the surface of the particles.

416

However, alginate coated CS nanoparticles could increase the stability of CS

417

nanoparticles in the PBS, resulting in extended release of antigen. The dissolution

418

data were plotted according to different models and drug release profiles of 13 Page 13 of 32

formulations F2 - F6 were compared with that of reference product (F1) to evaluate

420

the effect of process using the similarity factor (f2). The curvilinear nature of the

421

cumulative percent antigen released versus time plots depicts that the release of

422

antigen from the nanoparticles does not follow zero-order or first order kinetics

423

(Table 3). The in vitro dissolution studies suggest that drug release was governed by

424

Higuchi kinetics. However, the mathematical expression that best describes the

425

antigen release from these nanoparticles is the Makoid–Banakar model in which the

426

resultant R2 values were greater than 0.98. The Korsmeyer–Peppas release

427

exponent, n, is approximately 0.3, confirming that the release of antigen is governed

428

by a diffusion process. So it can be confirmed that antigen release is controlled by a

429

Fickian diffusion process that will also result in sustained release of antigen in blood.

430

Antigen release from nanoparticles was found to be affected by antigen-polymer

431

interaction, decreasing with higher MW of CS (Costa, & Sousa Lobo, 2003).

an

us

cr

ip t

419

432 Table 3

M

433 434

Fig. 2.

436

3.3.

Antigen released from uncoated and alginate coated CS nanoparticles in an

te

437

Stability study in simulated gastric fluid

d

435

acidic medium was analyzed by SDS-PAGE followed by Coomassie brilliant blue

439

staining. First The first prerequisite for effective oral vaccine formulation is to protect

440

antigen from the harsh conditions in the stomach after oral administration delivery.

441

According to Fig. 3, molecular weight marker was shown in Lane 6, and the measles

442

antigen incubated with PBS (pH 7.4) for 24 h exhibited three clear bands at about

443

80, 60 and 45 kDa (Lane 5). However, measles antigen pretreated with 0.01 M HCl

444

for 2 h had a faint smear (Lane 4) which indicated that the antigen underwent serious

445

degradation in an acidic medium.

446

Ac ce p

438

The SDS-PAGE data clearly indicates uncoated CS nanoparticles containing

447

measles antigen were degraded in the presence of acid. However, antigen from

448

alginate coated CS nanoparticles had clear bands at about 80, 60 and 45 kDa in

449

both the lanes (Lane 1 and Lane 2), which implied that alginate coating of CS

450

nanoparticles could effectively protect antigen from degradation in an acidic medium

451

for at least 2 h. So, the obtained alginate coated CS nanoparticles might be an

14 Page 14 of 32

452

effective oral antigenic carrier for mucosal vaccine. Keeping this in mind,

453

immunological study was performed only with alginate coated CS nanoparticles.

454 455

Fig. 3.

457

3.4.

458

ip t

456 In vitro cell viability studies

The MTT test results indicated that both alginate-coated (% cell viability 86.43 ± 4.60) and uncoated (% cell viability 82.58 ± 2.80) of comparatively higher MW of

460

CS (MW 106) nanoparticles of higher MW CS (MW 106) had minimal effects on HT-

461

29 cells even at higher concentration of 5 4 mg/ml. No significant difference in

462

cytotoxicity was observed among nanoparticles formulated with three different MW

463

(Fig. 4). These results are in good agreement with other studies where MW of CS did

464

not interfere with cell viability (Huang, Khor, & Lim, 2004; Opanasopit et al., 2007;

465

Richardson, Kolbe, & Duncan, 1999). Cytotoxicity was observed only for higher

466

concentration (5 4 mg/ml) for the formulation with CS of MW 42 kDa. Interestingly,

467

alginate coating increases cell viability, indicating that the cytotoxicity is related to the

468

charge density of the particle; toxicity increases with increasing nanoparticle positive

469

charge density (Yang, He, Duan, Kui, & Li, 2007).

470

472 473 474

Fig. 4.

Ac ce p

471

te

d

M

an

us

cr

459

3.5.

Immunological response

In the present study, we investigated the immunological response after the

475

administration of the measles antigen vaccine entrapped in alginate-coated CS LMW

476

nanoparticles was investigated. The humoral immune response was accompanied

477

by the presence of measles specific IgG and IgA in the serum and on the intestinal

478

mucosa, respectively. Sample collection for a preclinical DBS assay involves

479

spotting a small volume of blood (10-20 µl per spot) onto the special protein saver

480

card and drying subsequently to prepare DBS specimens. These cards consist of a

481

specialized matrix that lyses cells on contact, denatures proteins, and inactivates

482

bacteria and viruses. When required, a disc of 3 mm diameter is punched from this

483

card and the analytes are isolated by an elution buffer. A sensitive limiting dilution

484

analysis, comprising competitive and indirect ELISA, is carried out to quantitate the 15 Page 15 of 32

485

systemic humoral immune response and monitor the progress of antibody responses

486

in different groups of mice immunized with vaccine preparations.

487

The minimum dose of measles antigen dose (equivalent to 20 µg) was selected for the immunization study, calculated from the loading capacity of each

489

nanoparticle formulation, aimed at elucidating whether or not the MW of CS affects

490

systemic and mucosal responses. The protective anti-measles IgG titre value and

491

anti-measles IgA titre value results were found measured and results are plotted in

492

Fig. 5 and Fig. 6.

cr

ip t

488

493 3.5.1. Systemic antibody response

495

us

494

Mice were immunized orally with various formulations (Table 1) on days 0, 7, 14 and 28 and blood samples were drawn from the tail-vein on days 7, 14, 28, 56

497

post administration using a lancet. All the samples were tested in the same day with

498

same batch of reagents to minimize imprecision and bias. IgG titres were measured

499

in responder mice. The association of the measles antigen (group I-III) with the

500

alginate coated CS nanoparticles induced a strong humoral immune response that

501

was significantly higher (p < 0.05) than the mean titre found for group VI, vaccinated

502

with measles antigen orally (Fig. 5). When comparing statistically, the immunological

503

response elicited at each individual time point, the IgG levels for lower MW were

504

always significantly higher than those corresponding to the higher MW. With only

505

one boost, all the groups orally vaccinated with the antigen associated with coated

506

nanoparticles were able to show seroconversion. The formulations were found to

507

elicit responses that increased for 14 days and then levelled off. In fact, the group

508

vaccinated with the uncoated measles vaccine given orally showed a very low

509

responder number, whereas the group, with the measles antigen given

510

subcutaneously showed higher log antibody titre.

512

M

d

te

Ac ce p

511

an

496

Fig. 5.

513 514 515

3.5.2.

Mucosal anti-measles sIgA Mice were immunized orally with various formulations (Table 1) on days 0, 7,

516

14 and 28 and intestinal lavage fluids were collected on days 28 and 56 post

517

administration. The IgA anti-measles levels in the intestinal lavages indicate that oral

518

vaccination with alginate coated low MW CS nanoparticles induced stimulation of 16 Page 16 of 32

519

IgA-secreting cells of the GALT. There was a significant correlation between the IgA

520

levels produced by the nanoparticles vs. CS MW, similar to the case with humoral

521

response. In contrast, subcutaneous administration of formulation was not able to

522

induce any IgA response, not even after several booster immunizations (Fig. 6).

524

ip t

523 Fig. 6.

525

527

4.

Conclusion

cr

526

An effective vaccine formulation for oral delivery maintains the antigen in a stable form, protects antigen from enzymatic degradation and harsh conditions in the

529

stomach and intestine, adheres to the mucosal surface, ensures the antigen remains

530

in the gastro intestinal tract region long enough for the antigen to interact with the

531

lymphatic system, and efficiently stimulates innate responses, and evokes adaptive

532

immune responses that are appropriate for the target pathogen. The vaccine-loaded

533

alginate coated CS LMW nanoparticles could be prepared with suitable and

534

appropriate particle sizes, which is a very important factor in the delivery of the

535

vaccine to the induction site of mucosa-associated lymphoid tissue for proper

536

immune stimulation. Moreover, alginate coating onto the surface of nanoparticles

537

could modulate the release behaviour of the antigen and could effectively protect

538

antigen from degradation against acidic medium (pH 2) in vitro for at least 2 h. Both

539

systemic and local immune responses can be induced in a dose- and time-

540

dependent manner through vaccine-loaded CS nanoparticles. The data presented in

541

this study evince suggest that alginate-coated LMW CS nanoparticles can be used

542

widely for the oral delivery of therapeutics.

544 545 546

an

M

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te

Ac ce p

543

us

528

Acknowledgements

The authors thank Prof Debesh Chandra Majumder and Prof Pranabesh

Chakraborty for the encouragement and support provided.

547 548 549

References

550

Amoozgar, Z., Park, J., Lin, Q., & Yeo, Y. (2012). Low molecular-weight chitosan as

551

a pH-sensitive stealth coating for tumor-specific drug delivery. Molecular

552

Pharmaceutics, 9, 1262-1270. 17 Page 17 of 32

553

Anal, A. K., Bhopatkar, D., Tokura, S., Tamura, H., & Stevens, W. F. (2003).

554

Chitosan-alginate multilayer beads for gastric passage and controlled intestinal

555

release of protein. Drug Development and Industrial Pharmacy, 29, 713-724.

556

Baumann, U. (2008). Mucosal vaccination against bacterial respiratory infections.

558

Expert Review Vaccines, 7, 1257-1276.

ip t

557

Borges, O,. Cordeiro-da-Silva, A., Romeijn, S. G., Amidi, M., de Sousa, A., Borchard, G., & Junginger, H. E. (2006). Uptake studies in rat Peyer's patches,

560

cytotoxicity and release studies of alginate coated chitosan nanoparticles for

561

mucosal vaccination. Journal of Controlled Release, 114, 348-358.

cr

559

Borges, O., Silva, M., de Sousa, A., Borchard, G., Junginger, H. E., & Cordeiro-da-

563

Silva, A. (2008). Alginate coated chitosan nanoparticles are an effective

564

subcutaneous adjuvant for hepatitis B surface antigen. International

565

Immunopharmacology, 8, 1773-1780.

an

us

562

Borges, O., Tavares, J., de Sousa, A., Borchard, G., Junginger, H. E., & Cordeiro-

567

da-Silva, A. (2007). Evaluation of the immune response following a short oral

568

vaccination schedule with hepatitis B antigen encapsulated into alginate-coated

569

chitosan nanoparticles. European Journal of Pharmaceutical Sciences, 32, 278-

570

290.

d

M

566

Calvo, P., Remunan-Lopez, C., Vila-Jato, J. L., & Alonso, M. J. (1997). Chitosan and

572

chitosan/ethylene oxide-propylene oxide block copolymer nanoparticles as

573

novel carriers for proteins and vaccines. Pharmaceutical Research, 14, 1431-

Ac ce p

574

te

571

1436.

575

Chen, F., Zhang, Z. R., & Huang, Y. (2007). Evaluation and modification of N-

576

trimethyl chitosan chloride nanoparticles as protein carriers. International

577 578 579 580 581

Journal of Pharmaceutics, 336, 166-173.

Costa, P., & Sousa Lobo, J. M. (2003). Evaluation of mathematical models describing drug release from estradiol transdermal systems. Drug Development and Industrial Pharmacy, 29, 89-97. Csaba, N., Koping-Hoggard, M., & Alonso, M. J. (2009). Ionically crosslinked

582

chitosan/tripolyphosphate nanoparticles for oligonucleotide and plasmid DNA

583

delivery. International Journal of Pharmaceutics, 382, 205-214.

584 585

Czerkinsky, C., & Holmgren, J. (2009). Enteric vaccines for the developing world: a challenge for mucosal immunology. Mucosal Immunology, 2, 284-287.

18 Page 18 of 32

586 587

Delie, F., & Blanco-Prieto, M. J. Polymeric particulates to improve oral bioavailability of peptide drugs. Molecules, 10, 65-80. Du, C., Zhao, J., Fei, J., Gao, L., Cui, W., Yang, Y., & Li, J. (2013). Alginate-Based

589

Microcapsules with a Molecule Recognition Linker and Photosensitizer for the

590

Combined Cancer Treatment. Chemistry – An Asian Journal, 8, 736-742.

591

ip t

588

Eyles, J. E., Sharp, G. J., Williamson, E. D., Spiers, I. D., & Alpar, H. O. (1998). Intra nasal administration of poly-lactic acid microsphere co-encapsulated Yersinia

593

pestis subunits confers protection from pneumonic plague in the mouse.

594

Vaccine, 16, 698-707.

Fernandes, J. C., Qiu, X., Winnik, F. M., Benderdour, M., Zhang, X., Dai, K., & Shi,

us

595

cr

592

Q. (2012). Low molecular weight chitosan conjugated with folate for siRNA

597

delivery in vitro: optimization studies. International Journal of Nanomedicine, 7,

598

5833-5845.

600 601

Fooks, A. R. (2004). Development of oral vaccines for human use. Current Opinion in Molecular Therapeutics, 2, 80-86.

M

599

an

596

Gan, Q., Wang, T., Cochrane, C., & McCarron, P. (2005). Modulation of surface charge, particle size and morphological properties of chitosan-TPP

603

nanoparticles intended for gene delivery. Colloids and Surface B Biointerfaces,

604

44, 65-73.

607 608 609 610 611 612 613

te

606

Gazori, T., Khoshayand, M. R., Azizi, E., Yazdizade, P., Nomani, A., & Haririan, I. (2009). Evaluation of Alginate/Chitosan nanoparticles as antisense delivery

Ac ce p

605

d

602

vector: Formulation, optimization and in vitro characterization. Carbohydrate Polymers, 77, 599-606.

Godbey, W. T., Wu, K. K., & Mikos, A. G. (1999). Size matters: molecular weight affects the efficiency of poly(ethylenimine) as a gene delivery vehicle. Journal of Biomedical Material Research, 45, 268-275.

Hoff, J. (2000). Methods of blood collection in the lab mouse. Laboratory Animals, 29, 49–53.

614

Huang, M., Khor, E., & Lim, L. Y. (2004). Uptake and cytotoxicity of chitosan

615

molecules and nanoparticles: effects of molecular weight and degree of

616

deacetylation. Pharmaceutical Research, 21, 344-353.

617 618

Kim, B., Bowersock, T., Griebel, P., Kidane, A., Babiuk, L.A., Sanchez, M., AttahPoku, S., Kaushik, R. S., & Mutwiri, G. K. (2002). Mucosal immune responses

19 Page 19 of 32

619

following oral immunization with rotavirus antigens encapsulated in alginate

620

microspheres. Journal of Controlled Release, 85, 191-202. Kim, S. H., Lee, K. Y., & Jang, Y. S. (2012). Mucosal Immune System and M Cell-

622

targeting Strategies for Oral Mucosal Vaccination. Immune Network, 12, 165-

623

175.

ip t

621

Kostrzak, A., Cervantes Gonzalez, M., Guetard, D., Nagaraju, D. B., Wain-Hobson,

625

S., Tepfer, D., Pniewski, T., & Sala, M. (2009). Oral administration of low

626

doses of plant-based HBsAg induced antigen-specific IgAs and IgGs in mice,

627

without increasing levels of regulatory T cells. Vaccine, 27, 4798-4807.

Lavertu, M., Methot, S., Tran-Khanh, N., & Buschmann, M. D. (2006). High efficiency

us

628

cr

624

gene transfer using chitosan/DNA nanoparticles with specific combinations of

630

molecular weight and degree of deacetylation. Biomaterials 27: 4815-24

631

an

629

Lee, E., Lee, J., & Jon, S. (2010). A novel approach to oral delivery of insulin by conjugating with low molecular weight chitosan. Bioconjugate Chemistry, 21,

633

1720-1723.

M

632

Lee, S. J., Koo, H., Jeong, H., Huh, M. S., Choi, Y., Jeong, S. Y., Byun, Y., Choi, K.,

635

Kim, K., & Kwon. I. C. (2011). Comparative study of photosensitizer loaded and

636

conjugated glycol chitosan nanoparticles for cancer therapy. Journal of

637

Controlled Release, 152, 21-29.

640 641 642 643 644 645 646 647

te

639

Li. X., Kong. X., Shi. S., Zheng. X., Guo, G., Wei, Y. & Qian, Z. (2008). Preparation of alginate coated chitosan microparticles for vaccine delivery. BMC

Ac ce p

638

d

634

Biotechnology, 8, 89.

Liu, Z., Lv, D., Liu, S., Gong, J., Wang, D., Xiong, M., Chen, X., Xiang, R., & Tan, X. (2013). Alginic acid-coated chitosan nanoparticles loaded with legumain DNA vaccine: effect against breast cancer in mice. PLoS One 8: e60190

Malik, B., Goyal, A. K., Zakir, F., & Vyas, S. P. (2011). Surface engineered nanoparticles for oral immunization. Journal of Biomedical Nanotechnology, 7,

132-134. Mao, S., Shuai, X., Unger, F., Simon, M., Bi, D., & Kissel, T. (2004). The

648

depolymerization of chitosan: effects on physicochemical and biological

649

properties. International Journal of Pharmaceutics, 281, 45-54.

650

Mosmann, T. (1983). Rapid colorimetric assay for cellular growth and survival:

651

application to proliferation and cytotoxicity assays. Journal of Immunological

652

Methods, 65, 55-63. 20 Page 20 of 32

653 654

Neutra, M. R., & Kozlowski, P. A. (2006). Mucosal vaccines: the promise and the challenge. Nature Reviews Immunology, 6, 148-158. Oliveira, C. R., Rezende, C. M., Silva, M. R. Pego, A. P., Borges, O., & Goes, A. M.

656

(2012). A new strategy based on SmRho protein loaded chitosan nanoparticles

657

as a candidate oral vaccine against schistosomiasis. PLoS Neglected Tropical

658

Diseases, 6, e1894.

659

ip t

655

Opanasopit, P., Aumklad, P., Kowapradit, J., Ngawhiranpat, T., ApirakaraMWong, A., Rojanarata, T., & Puttipipatkhachorn, S. (2007). Effect of salt forms and

661

molecular weight of chitosans on in vitro permeability enhancement in intestinal

662

epithelial cells (Caco-2). Pharmaceutical Development and Technology, 12,

663

447-455.

us

cr

660

Prabakaran, M., Ho, H-T., Prabhu, N., Velumani, S., Szyporta, M., He, F., Chan, K-

665

P., Chen, L-M., Matsuoka, Y., Donis, R. O., & Kwang, J. (2009). Development

666

of Epitope-Blocking ELISA for Universal Detection of Antibodies to Human

667

H5N1 Influenza Viruses. PLoS ONE, 4, e4566.

M

an

664

Richardson, S. C., Kolbe, H. V., & Duncan, R. (1999). Potential of low molecular

669

mass chitosan as a DNA delivery system: biocompatibility, body distribution

670

and ability to complex and protect DNA. International Journal of Pharmaceutics,

671

178, 231-243.

674

te

673

Sergent, J. A., Paget, V., & Chevillard, S. (2012). Toxicity and genotoxicity of nanoSiO2 on human epithelial intestinal HT-29 cell line. The Annals of Occupational

Ac ce p

672

d

668

Hygiene, 56, 622-630.

675

Song, M. K., Vindurampulle, C. J., Capozzo, A. V., Ulmer, J., Polo, J. M., Pasetti, M.

676

F., Barry, E. M., & Levine, M. M. (2005). Characterization of immune responses

677 678

induced by intramuscular vaccination with DNA vaccines encoding measles virus hemagglutinin and/or fusion proteins. Journal of Virology, 79, 9854-9861.

679

van der Merwe, S. M., Verhoef, J. C., Verheijden, J. H., Kotze, A. F., Junginger, H.

680

E. (2004). Trimethylated chitosan as polymeric absorption enhancer for

681

improved peroral delivery of peptide drugs. European Journal of Pharmaceutics

682

and Biopharmacutics, 58, 225-235.

683

Vila, A., Sanchez, A., Janes, K., Behrens, I., Kissel, T., Vila Jato, J. L., Alonso, M. J.

684

(2004). Low molecular weight chitosan nanoparticles as new carriers for nasal

685

vaccine delivery in mice. European Journal of Pharmaceutics and

686

Biopharmaceutics, 57, 123-131. 21 Page 21 of 32

688 689 690 691

Wee, S., & Gombotz, W. R. (1998). Protein release from alginate matrices. Advanced Drug Delivery Reviews, 31, 267-285. Yang, Y., He, Q., Duan, L., Cui, Y., & Li, J. (2007). Assembled alginate/chitosan nanotubes for biological application. Biomaterials, 28, 3083-3090. Yoo, M. K., Kang, S. K., Choi, J. H., Park, I. K., Na, H. S., Lee, H. C., Kim, E. B.,

ip t

687

Lee, N. K., Nah, J. W., Choi, Y. J., & Cho, C. S. (2010). Targeted delivery of

693

chitosan nanoparticles to Peyer's patch using M cell-homing peptide selected

694

by phage display technique. Biomaterials, 31, 7738-7747.

695

cr

692

Zaharoff, D. A., Rogers, C. J., Hance, K. W., Schlom, J., & Greiner, J. W. (2007). Chitosan solution enhances both humoral and cell-mediated immune

697

responses to subcutaneous vaccination. Vaccine, 25, 2085-2094.

699

Zhu, Q., & Berzofsky, J. A. (2013). Oral vaccines: directed safe passage to the front

an

698

us

696

line of defense. Gut Microbes, 4, 246-252.

List of abbreviations used: PAGE- polyacrylamide gel electrophoresis, GALT-gut

701

associated lymphoid tissue

M

700

702

te Ac ce p

704

d

703

22 Page 22 of 32

Fig. 1. Scanning electron microscopy image of antigen loaded alginate coated CS

705

(MW 106 kDa) nanoparticle after freeze-drying process, magnification 6000X.

706

Fig. 2. In vitro release profiles of antigen from uncoated and alginate coated CS

707

nanoparticles in PBS at 37 OC in the first 120 h.

708

Fig. 3. Gel electrophoresis analysis, obtained under conditions of alginate coated

709

(Lane 1) and uncoated (Lane 2) CS (MW 106 kDa) nanoparticles, alginate coated

710

(Lane 3) CS (MW 42 kDa) nanoparticles containing antigen pre-treated with 0.01 M

711

HCl for 2 h, then sustained release in PBS for 24 h. Measles antigen pre-treated with

712

0.01 M HCl for 2 h, then incubation with PBS for another 24 h (Lane 4), measles

713

antigen incubation with PBS for 24 h (Lane 5), Molecular weight marker (Lane 6).

714

Fig. 4. Effect of (A) uncoated and (B) alginate coated CS nanoparticles at different

715

concentrations on the viability of HT 29 cells determined by MTT assay. Indicated

716

values were mean ± SD (n = 6). Statistical difference between the control groups and

717

formulations are reported as * p < 0.05, no significant difference, ns p > 0.05.

718

Fig. 5. Anti-measles IgG titres of mice immunized with different alginate-coated CS

719

LMW nanoparticle oral formulations of measles vaccine. Values are expressed as

720

antibody titres of mice on day 7, 14, 28, 56 post administration. Mean reciprocal

721

dilutions are represented as the end point titre (log10) ± SD. Titres were defined as

722

the highest dilution resulting in an absorbance value twice that of non-immune

723

plasma.

724

Fig. 6. Secretory anti measles sIgA profile after oral administration of various

725

formulations in CS LMW nanoparticles detected in individual intestine washing

726

samples of mice giving an OD of 0.1 after subtraction of background and presented

727

as geometric mean titre per group.

729 730

cr

us

an

M

d

te

Ac ce p

728

ip t

704

731

23 Page 23 of 32

731

Table 1. Measles antigen loaded vaccine formulation for immunization study

732 Table 2. Particle size, zeta potential and loading capacity of antigen loaded three

734

different molecular weight of CS-LMW nanoparticles without or with alginate coated

735

nanoparticles (n = 3, mean ± SD)

ip t

733

736

Table 3. Results of model fitting of antigen release of various nanoparticles in

738

phosphate buffer pH 7.4

cr

737

739

Table 1. Measles antigen loaded vaccine formulation for immunization study Formulation

us

740

Description

Group

AL-CS-42-MA

Measles

an

code antigen

loaded

alginate I

coated CS (MW 42 kDa) nanoparticles Measles

antigen

loaded

M

AL-CS-74-MA

alginate II

coated CS (MW 74 kDa) nanoparticles Measles

antigen

coated

CS

loaded

(MW

d

AL-CS-106-MA

106

alginate III kDa)

AL-CS-42

te

nanoparticles

Alginate coated CS (MW 42 kDa) IV

Ac ce p

nanoparticles, used as negative control

MA-SC

Measles

subcutaneously,

antigen used

given V as

positive

control

MA-ORAL 741 742

Measles antigen given orally

VI

24 Page 24 of 32

742

Table 2. Particle size, zeta potential and loading capacity of antigen loaded three

743

different molecular weight of CS-LMW nanoparticles without or with alginate coated

744

nanoparticles (n=3, mean ± SD) Mean particle Zeta

Composition

size (nm)

LMW-CS (42 kDa)- 284 ± 36

cr

Alginate/LMW-CS

432 ± 52

+1.65 ± 0.4 0.368 ± 0.06

LMW-CS (74 kDa)- 507 ± 37

+21.4 ± 1.2 0.412 ± 0.04

us

(42 kDa)-Antigen 3

+14.2 ± 0.6 0.472 ± 0.05

an

2.50 ± 0.32

Alginate/LMW-CS

1003 ± 45

-24.1 ± 0.9

0.484 ± 0.03

1.23 ± 0.18

(106 kDa)-Antigen

-10.1 ± 0.6

(106

M

LMW-CS

te

746

2.10 ± 0.26

Ac ce p

745

3.41 ± 0.88

852 ± 76

651 ± 28

kDa)-Antigen 6

1.56 ± 0.14

0.438 ± 0.08

Alginate/LMW-CS (74 kDa)-Antigen

5

2.87 ± 0.46

d

Antigen 4

capacity (%)

+31.6 ± 1.4 0.310 ± 0.08

Antigen 2

Loading

potential (mV)

1

PDI

ip t

S. No.

25 Page 25 of 32

747

phosphate buffer pH 7.4 Para

Formulation code

model

meter

NP-CS-

NP-CS-

NP-CS-

NP-AL-

NP-AL-

NP-AL-

42

74

106

CS-42

CS-74

CS-106

0.684

0.698

0.742

0.664

0.689

0.631

0.011

0.009

0.008

0.006

0.005

0.004

0.915

0.928

0.952

0.927

6.390

5.725

5.044

4.214

3.666

3.017

0.991

0.986

0.987

0.982

0.986

0.989

12.846

10.889

8.684

7.967

6.527

5.632

0.338

0.350

0.374

0.352

0.366

0.355

0.994

0.995

0.996

0.997

0.995

0.994

13.809

12.454

10.123

9.526

7.631

6.225

0.272

0.289

0.250

0.280

0.297

-0.002

-0.003

-0.003

-0.002

51.14

41.07

36.28

31.83

Korsmey erPeppas

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Banakar

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0.295

-0.001

-0.002

Referen 66.05

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0.949

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Kinetic

M

Table 3. Results of model fitting of antigen release of various nanoparticles in

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748 749

26 Page 26 of 32

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