[Methods in Enzymology] Nanomedicine - Infectious Diseases, Immunotherapy, Diagnostics, Antifibrotics, Toxicology and Gene Medicine Volume 509 || Chitosan-Based Nanoparticles as a

C H A P T E R S E V E N

M

IS

*{

ethods

SN 0

CentFaculCoim

Chitosan-Based Nanoparticles as a

Hepatitis B Antigen Delivery System

Filipa Lebre,*,† Dulce Bento,*,† Sandra Jesus,*,† and

Olga Borges*,†

Contents

1. In

in

076

er foty obra

troduction

Enzymology, Volume 509 # 2012

-6879, DOI: 10.1016/B978-0-12-391858-1.00007-1 All rig

r Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugalf Pharmacy, University of Coimbra, Polo das Ciencias da Saude Azinhaga de Sa, Portugal

Else

hts

nta

128

2. C
hitosan-Based Particle Preparation 129
2
.1. A lginate-coated chitosan particles 129
2
.2. C hitosan nanoparticles 130
2
.3. C hitosan/alginate particles 130
2
.4. C hitosan/poly-e-caprolactone particles 131
3. P
hysicochemical Characterization of the Particles 132
3
.1. S ize measurement 132
3
.2. Z eta potential titration 133
3
.3. S canning electron microscopy 133
4. A
ntigen Adsorption Studies 134
5. In
vitro Release Studies 135
6. E
valuation of the Bioactivity of the Antigen 137
7. C
ell Viability Studies with Spleen Cells 137
7
.1. P reparation of spleen cell suspensions 138
7
.2. M TT viability assay 138
8. S
tudies on Uptake into Peyer’s Patches 139
9. C
oncluding Remarks 140
Refe
rences 140
Abstract

The design of antigen delivery systems, particularly for mucosal surfaces, has

been a focus of interest in recent years. In this chapter, we describe the

preparation of chitosan-based particles as promising antigen delivery systems

for mucosal surfaces already tested by our group with hepatitis B surface

antigen. The final proof of the concept is always carried out with immunization

studies performed in an appropriate animal model. However, before these

vier Inc.

reserved.

Comba,

127

128 Filipa Lebre et al.

important studies, it is advisable that the delivery system should be submitted

to a variety of in vitro tests. Among several tests, the characterization of the

particles (size, morphology, and zeta potential), the studies of antigen adsorp-

tion onto particles, the evaluation of toxicity of the particles, and the studies

of particle uptake into lymphoid organs are the most important and will be

described in this chapter.

1. Introduction

At present, the term “vaccination” is generally considered to beidentical to “injection.” This conception is due to the fact that vaccinesare typically given by intramuscular injection. In the new era of vaccinedevelopment, with the emergence of subunit vaccines, the formulation ofneedle-free vaccines is undoubtedly more challenging. Novel vaccinesobtained by recombinant technology are, in principle, safer with regard totoxicity; however, they are also less immunogenic, making it mandatory toinclude adjuvants in the formulation of such vaccines. Numerous effortsmade by the scientific community to develop needle-free vaccine formula-tions are justifiable by several distinct advantages. An obvious one is thepossibility of painless self-administration of the vaccine. Moreover, vaccinedelivery via mucosal surfaces elicits mucosal immune responses at the site ofpathogen entry and enhances cellular immunity through stimulation ofToll-like receptors (Bessa and Bachmann, 2010), thus improving overalleffectiveness. Taking these facts into account, needle-free vaccination couldhave a big impact on the efficacy of immunization against mucosal trans-mitted diseases such as hepatitis B. In 1981, FDA approved the first hepatitisB vaccine which consisted of the surface antigen of the hepatitis B (HBsAg)virus present in the blood of human carriers of the infection, replaced in1986 by the currently available vaccine which represents the world’s firstsubunit vaccine and the world’s first recombinant expressed vaccine. Sincethe hepatitis B virus can be transmitted perinatally or by exchange of bodyfluids (e.g., blood, semen, and vaginal fluid), the design of new hepatitisB vaccines with the additional possibility to induce mucosal antibodies(e.g., secretory IgA) is particularly attractive. The only available hepatitisB vaccines to date are injectable formulations, adjuvanted with aluminumsalts, which are evidently not appropriate for oral or intranasal administra-tion owing to two main reasons. One, mucosally administered antigens willbe exposed to enzymatic degradation, and second, the adjuvant is notadequate for application at mucosal surfaces. Therefore, formulations withenhanced adjuvant properties are needed for the application at mucosalsurfaces to reduce the high antigen doses normally required to increasethe low immune response and decrease the variability of the individual

Chitosan-Based Nanoparticles as a Hepatitis B Antigen Delivery System 129

immune responses frequently observed. Preclinical investigation of newneedle-free hepatitis B vaccines relies on the development of adjuvants/new formulations with additional capability to increase the immunogenicityof the antigen. To achieve this goal, several strategies are currently beingdiscussed (Lebre et al., 2011; Thanavala et al., 2009). A good example is thedevelopment of nanosized carrier systems that adsorb or encapsulate anti-gens, protect them from proteolytic enzymes, allow the increase of antigenretention time at the nasal mucosa, and finally target antigens to M-cellspresent on the mucosa ( Jabbal-Gill, 2010). Lastly, the loading of particlesnot only with antigens but also with immunopotentiators such as combina-tions of Toll-like receptor ligands (Kasturi et al., 2011) may modulate thequantity and the quality of the immune response.

Chitosan is a cationic polymer consisting of b-(1-4)-linked D-glucosamine(deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit) mono-mers that can be obtained by deacetylation of chitin (Illum, 1998). It hasbeen considered a nontoxic, biodegradable, and biocompatible polymer(Baldrick, 2010), thus extensive research has been directed toward its usein medical applications such as drug and vaccine delivery (Lebre et al., 2011;Panos et al., 2008; van der Lubben et al., 2001b). One major advantage ofthis polymer is its ability to easily produce nanoparticles under mildconditions without the application of harmful organic solvents. This hasbeen one of the main reasons for its wide applicability to the encapsulationof different molecules such as therapeutic proteins, DNA, and antigens.Chitosan is also known to be mucoadhesive, and its ability to stimulate cellsof the immune system has been shown in many studies (Borges et al.,2007a). These unique features make chitosan an attractive polymer to actas an adjuvant.

2. Chitosan-Based Particle Preparation

2.1. Alginate-coated chitosan particles

Chitosan nanoparticle preparation can be achieved by several techniques.One of the most common is the precipitation/coacervation method, whichis a process of spontaneous phase separation that occurs when two oppo-sitely charged polyelectrolytes are mixed in an aqueous solution.

The protocol used in our laboratory results from an adaptation andoptimization of a previously described method (Berthold et al., 1996).The preparation of this delivery system contains three main steps:manufacturing of the chitosan particles, their loading by adsorption, andfinally coating with sodium alginate (Borges et al., 2005). Low molecularweight chitosan is dissolved at a concentration of 0.25% (w/v) in a solutionwith 2% (v/v) of acetic acid and 1% (w/v) of Tween

TM

80. The formation of


the particles is achieved after the addition of 3.5 ml of sodium sulfatesolution (10%, w/v) to 200 ml of the chitosan solution. The addition ismade at a rate of 1 ml/min under mild agitation (<50 rpm) and continuoussonication (VibraCell sonicator, 600-watt model; Sonics & Materials, Inc.,Newtown, CT, USA). Sonication is maintained for an additional 15 minand the agitation for 60 min at room temperature (RT). The suspension iscentrifuged for 30 min at 2800� g and the supernatant is discarded. Theparticles are resuspended twice in Milli-Q water, centrifuged again for30 min, and the supernatants are discarded. The particles are frozen in liquidnitrogen and freeze–dried overnight using a freeze-dryer. The dry powderis kept frozen until further use.

At this point, particles can be loaded with proteins of biological interest.In our case, we loaded them with HBsAg so they can act as a delivery systemfor the antigen. Loading studies will be described later in the chapter. Inorder to ensure the stability and protection of the antigen, loaded particlesare coated with sodium alginate. For this purpose, equal volumes of theantigen-loaded nanoparticle suspension and a buffer phosphate solution ofsodium alginate (1%, w/v) are mixed under magnetic stirring. The agitationis maintained during a 20-min period. The suspension is then centrifugedfor 10 min at 460� g and the supernatant is discarded. The particles areresuspended in 0.524 mM CaCl2 in 50 mM HEPES buffer solution and keptunder agitation for another 10 min. A laboratory temperature below 20 �C iscrucial for these experiments.

2.2. Chitosan nanoparticles

A second protocol used in our laboratory results from an adaptation of amethod previously described (Roy et al., 1999). Briefly, equal volumes ofa solution of chitosan (0.1% in sodium acetate buffer, 25 mM, pH 5.0) and asodium sulfate solution (0.625%) are mixed under high-speed vortexing for20 s. The resultant nanoparticles are left to rest at RT for approximately 1 h.In order to remove compounds that did not react, the resulting nanoparticlesuspension is centrifuged for 30 min at 4500� g. The supernatant is discardedand the obtained pellet resuspended in sodium acetate buffer, 25 mM, pH5.5. Nanoparticles should be used immediately after resuspension to avoidparticle aggregation.

2.3. Chitosan/alginate particles

Alginate is a biodegradable and a biocompatible natural polyanionic poly-saccharide with a good safety profile. Its molecular structure consists oflinear copolymers of L-guluronic and D-mannuronic acid residues joinedlinearly by 1,4-glycosidic linkages. Divalent cations such Ca2þ, Ba2þ, andSr2þ work as alginate cross-link agents inducing gel formation via a sol–gel


transformation (Wee and Gombotz, 1998). Calcium ions have higheraffinity to guluronic acid residues, so the relative composition of the alginatecan have impact in characteristics of the delivery system. Therefore, algi-nates with higher guluronic acid content tend to form more rigid structureand higher porosity than alginates rich in mannuronic acids (De andRobinson, 2003).

Chitosan/alginate (Chi/Alg) particles are prepared using a two-stepmethod modified from Rajaonarivony et al. (1993). In order to preparethe pregel, 3 ml of a calcium chloride solution (2 mg/ml) is added dropwiseto 47 ml of sodium alginate solution 0.063% (pH 5.1) in an ultrasound bathwhile stirring for 15 min at 25,000 rpm with a homogenizer (Ystral GmbH,Dottingen). Ca2þ/alginate pregel is stirred for another 20 min with a mag-netic stirrer. Finally, particles are formed upon mixing 1.5 ml of pregel withan equal volume of chitosan 0.1% (acetic acid solution; pH 5.4) under high-speed vortexing following additional 30 min of magnetic stirring allowingnanoparticle maturation. Nanoparticles are isolated by centrifugation at5000� g for 40 min at 20 �C. The supernatant is discarded and the pelletresuspended in the intended buffer (e.g., phosphate buffer (PB), pH 7.4 forprotein adsorption studies).

2.4. Chitosan/poly-e-caprolactone particles

As we have discussed above, chitosan nanoparticles offer some advantages asdrug delivery systems. Nevertheless, the inclusion of a hydrophobic poly-mer like poly-e-caprolactone (PCL) might confer additional useful proper-ties to the delivery system, like the possibility to establish hydrophobicinteractions between delivery system and loaded proteins.

The procedure for the production of chitosan/PCL particles in ourlaboratory resulted from the adaptation of and experimentation of differenttechniques described in the literature, in particular, one described byBilensoy et al. (2009) that is based on the nanoprecipitation techniquepatented by Fessi et al. (1992). An aqueous phase of 0.1% acetic acidcontaining 0.1% chitosan and 5% Tween

TM

80 is placed under high-speedhomogenization. Then, the organic phase, consisting of 0.2% PCL dilutedon acetone, is added dropwise to the first solution at a proportion of 1:3(v/v) to a final volume of 18 ml. The resultant particle suspension is placedunder magnetic stirring for additional 45 min for the maturation process.Finally, the organic phase is removed by evaporating acetone with a nitro-gen flux in a warm bath (40 �Cmaximum). The nanoparticles suspended inthe original medium can be isolated, concentrated, and resuspended in otherdiluents by centrifugation at 16,000� g, for 75 min at 4 �C. To guaran-tee minimal aggregation of the particles during the centrifugation, a 200 mlglycerol bed for each 18 ml batch is recommended.


3. Physicochemical Characterization

of the Particles

3.1. Size measurement

It is generally accepted that the size and size distribution of the particles areimportant for their adjuvant activity. Therefore, size characterization is animportant step in vaccine formulation development even if the attempts tocorrelate particle size and the resultant immune responses lead to conflictingfindings (Oyewumi et al., 2010).

The size of particles can be measured by Dynamic Light Scatteringtechniques. Among those techniques, Photon Correlation Spectroscopy(PCS) has been widely used as routine standard technique in biophysics,colloid and polymer laboratories. PCS is based on the fact that the intensityof light scattered from a dispersion of particles into a given scattering angle isthe result of interference on the surface of a square-law detector betweenlight scattered from different particles in the medium (Pecora, 2000). PCSgives the translational self-diffusion coefficient of the nanoparticle (Pecora,2000), which, for particles in a dilute dispersion, can be related withhydrodynamic diameter of the particle (nonspherical or flexible particle)through the Stokes–Einstein equation (Eq. 7.1):

d Hð Þ¼ kT

3p�Dð7:1Þ

d(H), hydrodynamic diameter; D, translational diffusion coefficient; k,Boltzmann’s constant; T, absolute temperature; �, viscosity.

Additionally, particles in suspension undergo Brownian motion due torandom bombardment by the solvent molecules that surround them. Whenparticles are illuminated with a laser, the intensity of the scattered lightfluctuates at a rate dependent of the size of the particles (Malvern, 2004).The smaller particles move quickly and induce the intensity to fluctuatemore rapidly than the larger ones. Thus, the analysis of the rate of intensityfluctuations using the autocorrelation function allows the determination ofthe particle size distribution of the sample.

It is important to be aware that PCS measures the hydrodynamicdiameter, which refers to how a particle diffuses within a fluid and corre-sponds to the diameter of a sphere that has the same translational diffusioncoefficient as the particle that is being measured. The size of the particle“core” is not the only determinant of the translational diffusion coefficient.Thus, factors like ionic strength of the medium and surface structures thatcan affect the particles’ diffusion speed will possibly change the apparent sizeof the particle. Samples for PCS analysis should consist of a well-dispersed


phase in a suspending medium, and both the refractive index of the solventand the viscosity at the selected measurement temperature must be known.

3.2. Zeta potential titration

In a colloidal system, when a particle is dispersed in a fluid, a range ofprocesses causes the interface to become electrically charged. The liquidlayer surrounding the particle can be divided into two parts: the innerregion where ions are strongly bound and the outer region where theyare less firmly associated to particle. On this outer region named the diffuselayer, there is a notional boundary inside which the ions and particles form astable entity. The potential at this boundary is the zeta potential. Althoughzeta potential occurs at a distance from the particle, it is related to it andtherefore influences a wide range of properties of colloidal systems, such asstability, interaction with electrolytes, and suspension rheology.

Theoretically, nanoparticles with a zeta potential above (þ/�) 30 mVhave been shown to be stable in suspension, as the surface charge preventsaggregation of the particles. Several researchers have been shown that thestability of the nanoparticles is highly dependent on the pH values, andoptimal pH value can result in the highest stability of the nanoparticles. Theother important parameter is the ionic strength of the medium, when it ishigh, zeta potential becomes closer to zero. Electrostatic repulsion disap-pears due to an increase of salt concentration which compresses the electri-cal field double layer of the particles.

The pH of the medium is one of the most important factors that affectzeta potential. When a particle is in suspension and alkali is added to thesuspension, the particles will acquire negative charges, decreasing its zetapotential and on its turn, if acid is added, particles will acquire positivecharges, increasing its zeta potential. Considering this phenomenon, we canperform a zeta potential titration to characterize nanoparticles, once thisvariable is highly dependent on the conditions of the suspending medium.The results can be expressed on a graph like the one represented in Fig. 7.1.Measuring zeta potential variation at different pHs, we can observe positivevalues at low pH and lower or negative values at high pH. The point wherethe plot passes through zero is the isoelectric point of the particles, normallythe point where the colloidal system is less stable. In a very simplistic way,we can say that two pH values above or below the isoelectric point, thecolloidal suspension starts to be stable (Malvern, 2004).

3.3. Scanning electron microscopy

Size and morphology of nanoparticles can be observed by scanning electronmicroscopy (SEM) using an electron microscope such as JSM-700 1FA (JOEL, Japan). Prior to image acquisition, one drop of nanoparticle

Zeta potential titration

3 6 9 12

-60

-40

-20

0

20

40

pH

Zeta

pot

entia

l (m

V)

Figure 7.1 An example of zeta potential titration of chitosan/PCL nanoparticles.Measurements of the nanoparticle zeta potential were performed on the Delsa

TM

Nano C, by resuspending 100 ml of a nanoparticle suspension on 1.9 ml of an acidic orbasic solution.


suspension is placed over a copper surface and let to dry overnight. After-ward, samples are mounted on microscope stub, coated with gold, and thenobserved on microscopy. Figure 7.2 represents SEM images of chitosanparticles prepared in our laboratory. It is frequently observed that during thedrying, the particles tend to stick together.

4. Antigen Adsorption Studies

Polymeric particles can be used as delivery systems for molecules withbiological interest such as proteins or more specifically antigens. The adsorp-tion of the antigens onto particles is a mild process since can be performedsimply by the incubation of the particles with the solution of the antigens atRT. Furthermore, given that the antigens are located at the surface of theparticles, it is expected that they are more available to be presented byantigen-presenting cells. However, protein adsorption is a complex processthat is affected by a number of factors concerning protein (charge, size, andstructure), polymer (size, composition, hydrophobicity, and zeta potential),and medium properties (pH, ionic strength, and viscosity) (Gonzalez Ferreiroet al., 2002; Kim et al., 2002). The adsorption of a protein onto the hydro-philic chitosan particles is mainly caused by electrostatic interaction of theprotonated chitosan amino groups with the carboxyl groups of the proteinsubstrate in a buffer. It is prudent to start the adsorption studies with modelantigens which are less expensive than the real antigens to obtain preliminaryinformation. According to the objective of the assay, these studies are per-formed suspending the particles into a buffer solution (PB) with the modelantigen. In order to investigate the best conditions that generate the highestparticle loading capacity (LC) and antigen loading efficacy (LE), different

Figure 7.2 SEM image of chitosan nanoparticles (JEOL JSM 6400 scanning electronmicroscopy) revealed the presence of small rough rounded particles (�100 nm).


concentrations of the antigens should be experimented at a fixed time andfixed particle concentration (see results on Fig. 7.3). After incubation, aliquotsof the particle suspension are centrifuged at 18,000� g for 30 min and super-natant is collected to measure nonbound protein (indirect method) and deter-mine the LE (Eq. 7.2) and the LC (Eq. 7.3):

LE %ð Þ¼ total amount of protein�nonbound proteinð Þtotal amount of protein

�100 ð7:2Þ

LC %ð Þ¼ total amount of protein�nonbound proteinð Þweight of the particles

�100 ð7:3Þ

5. In vitro Release Studies

To evaluate the suitability of nanoparticles as a delivery system forproteins with biological interest such as the HBsAg, an in vitro release studyshould be performed. These studies are performed with different buffers

Nanoparticle loading capacity — comparison of the delivery systems

Ovalbumin140

α-Casein160 c

BSA160 c

100

120c

120

140a 120

140

60

8080

10080

100

40

a

LC

(%

)

LC

(%

)

LC

(%

)

40

604060

a

0

20b

0

20b

020 b

Chi Chi/Alg Chi/PCL Chi Chi/Alg Chi/PCL Chi Chi/Alg Chi/PCL

Lysozyme100

Myoglobin120

b

Lactalbumin100

80b 80

100 80c

6060

60

20

40a

a

LC

(%

)

20

4040

a

LC

(%

)

LC

(%

)

20 b

a

Chi Chi/Alg Chi/PCL0

Chi Chi/Alg Chi/PCL Chi Chi/Alg Chi/PCL0

a0

Chi/Alg Chi/PCLihCSize (nm) 592.0 ± 25.6 3613.9 ± 1655.6 169.0 ± 7.4

Zeta potential (mV) + 5.50 ± 0.55 –15.74 ± 2.17 –8.18 ± 1.77

Figure 7.3 Comparison of the particle’s loading capacity (LC) using different proteins.The results illustrated on this figure were obtained during adsorption studies performedwith buffer phosphate, pH 7.1�0.2 with six proteins with different isoelectric points.Chi/PCL nanoparticles have a significantly higher LC when compared with Chi andChi/Alg particles for all the proteins except lysozyme. The inclusion of a hydrophobicpolymer like PCL into chitosan particles allows the establishment of hydrophobicinteractions between proteins and particles, which explains, at least in part, the result.In contrast, Chi/Alg particles have the lowest LC for almost all the proteins. Theadsorption of proteins with low IEP (<7.0) is below 20% and about 60% for lysozyme(IEP�11.0). This last result can be explained by the negative charge of particles in PB7.4 which favors electrostatic interactions only with the positively charged proteins.The table below the chart illustrates the size and zeta potential of chitosan-baseddelivery systems suspended in phosphate buffer, pH 7.4 (mean�SD, n¼3), duringadsorption studies.


which mimetize the physiological conditions. To mimic the digestive tract,simulated gastric fluid and simulated intestinal fluid, both described by UnitedStates Pharmacopeia, are normally used as release buffers, by our group tostudy oral antigen delivery systems. The antigen delivery systems developedto be administered by nasal or by one of the parenteral routes of theadministration should be tested with PB or even better with phosphate-buffered saline (PBS), pH 7.4. For the release studies, aliquots of the anti-gen-loaded nanoparticle suspension are added to individual tubes containingrelease medium previously equilibrated at 37 �C and placed in a shaker bathadjusted to 50 rpm. At appropriate time intervals, samples from each tube arecollected and filtered with a low protein-binding filter (MILLEXÒGV—0.22 mm; durapore PVDFmembrane;Millipore,Molsheim, France) followed


by centrifugation for 20 min at 18,000� g and the protein in supernatantassayed with an appropriated method. Suspensions of unloaded particles alsohave to be analyzed under the same conditions to evaluate possible interfer-ences on the protein quantification method.

6. Evaluation of the Bioactivity of the Antigen

The preliminary evaluation of the bioactivity of the antigen, after theirassociation with particles, is normally performed using the Western blottingtechnique. The integrity and bioactivity of hepatitis B antigen are done afterantigen being released from nanoparticles. Therefore, the samples resultingfrom the release studies mentioned above need to be centrifuged at 14,000rpm in order to separate the released antigen from the particles and then analiquot is solubilized with the SDS-PAGE loading buffer and treated 5 min at100 �C. The SDS-PAGE is performed in accordance with standard protocols(Gallagher and Smith, 2003) with 12% resolving gel, cast and run in Tris–glycine buffer at 25 mA. The antigenicity of the entrapped hepatitis B antigenis assessed by immunoblotting using a mouse antiserum raised against thenative antigen. The hepatitis B antigen samples were transferred from theunstained gel onto a nitrocellulose membrane, using a semi-dry electroblot-ting system (115 mA; 1 h) and the membrane is blocked overnight at 4 �Cwith PBS-T (0.05% of Tween 20) containing 5% of milk. After washing withPBS-T, the membrane is incubated for 2 h at RT with the positive anti-HBsAg IgG mouse antiserum, diluted 1:500 in PBS-T with 5% of low-fatmilk. After washing with PBS-T, the membrane is incubated with antimouseIgG conjugated to alkaline phosphatase, diluted 1:750. The ability of themouse antiserum to recognize hepatitis B antigen released from the nano-particles is demonstrated colorimetrically using 5 ml of phosphatase bufferwith 33 ml NBT (50 mg/ml) and 16.7 ml BCIP (50 mg/ml). The reaction isstopped by washing the membrane with water (Borges et al., 2007b). It isexpected that the HBs-specific antibodies recognize the antigen epitopesfrom the sample in a similar way as for the original antigen. With this analysis,it is then possible to confirm if the antigenicity of the hepatitis B antigen isaltered after antigen adsorption with nanoparticles which can compromise theefficacy of the formulation developed (Borges et al., 2007b).

7. Cell Viability Studies with Spleen Cells

There are different methods for assessing the in vitro cytotoxicity.Alterations in plasma membrane permeability can be evaluated both bythe release of cytoplasmic enzymes (e.g., lactate dehydrogenase) or by the


uptake of dyes (e.g., trypan blue, propidium iodide). Alternatively, cyto-toxicity can be evaluated by changes in cell metabolic activity. Tetrazoliumsalts are widely used in these metabolic assays (e.g., MTT, XTT, WST-1).In our laboratory, the MTT assay (Sigma-Aldrich, St. Louis, MO, USA) isnormally used to evaluate the cytotoxicity of the particles. In this assay, thetetrazolium salt is reduced to purple formazan crystals by metabolicallyactive cells. The principal reason for the choice of spleen cells to assess thecytotoxicity of nanoparticles intended for mucosal immunization is relatedto the fact that they are a very good and sensitive representative of thedifferent immune cells and are obtained and cultured easier, compared toother lymphoid organs, like Peyer’s patches (Borges et al., 2006).

7.1. Preparation of spleen cell suspensions

Mice are euthanized by cervical dislocation and their spleens are asepticallyremoved. Individual spleen cell suspensions are prepared in a Petri dishusing curved needles. One needle is used to hold the spleen and the other todetach cells from the capsule by moving the needle along the length of thespleen. The cell suspension is then transferred into a 15-ml sterile conicaltube to allow large fragments to settle down for 5 min. The cell suspension isdecanted into another sterile centrifuge tube and is centrifuged for 10 min at259� g. The resultant supernatant is discarded and cells are resuspended in5 ml of RPMI 1640. This washing step is repeated two times, and finally, thecells are resuspended in complete RPMI 1640 medium (supplemented with10% (v/v) fetal bovine serum, 1% (v/v) glutamine, 1% (v/v) gentamicin,and 2% (v/v) 1M HEPES buffer). The final suspension is adjusted to a finalconcentration of 5�106 cells per ml.

7.2. MTT viability assay

One-hundred microliters of aseptically prepared nanoparticles are resus-pended in complete RPMI and platted in a 96-well plate. One-hundredmicroliters of spleen cell suspension (5�105 cells/well) is then added to thewells. Cell and particles are incubated for 24 h (95% relative humidity and 5%CO2.) at 37

�C. MTT solution (5 mg/ml in PBS, pH 7.4) is filtered toremove any precipitate (0.22-mm filter), preheated at 37 �C, and added toeach well (20 ml/well). The plate is then incubated for additional 4 h. In theend, the plate is centrifuged for 25 min (800� g) and supernatant removedusing a multichannel pipette. To dissolve the formazan crystals, 200 ml ofpreheated (37 �C) DMSO are added to each well and pipetted up and down(carefully, to avoid any bubble formation). The plate is mixed in a plate shakerfor 10 min and incubated at 37 �C for 30 min. After the incubation time,optical density (OD) of plate solutions is read at 540 nm with 630 nm aswavelength reference. The relative cell viability (%) related to control wells


containing spleen cells in culture medium without nanoparticles is calculatedby Eq. (7.4):

cell viability %ð Þ¼ OD sample ð540nmÞ�OD sample 630nmð ÞOD control 540nmð Þ�OD control 630nmð Þ�100

ð7:4Þ

It is important to notice that this protocol is optimized for spleen cells.For a different cell line, the linear relationship between metabolically activecell number and signal produced (color), as well as the incubation time,should be established, allowing an accurate quantification of cell viability.

8. Studies on Uptake into Peyer’s Patches

Oral vaccination presents advantages over parenteral injection, never-theless the degradation of the vaccine and the low uptake by the gut-associated lymphoid tissue are determinant factors that limit the success ofthis strategy ( Jung et al., 2000; Van Der Lubben et al., 2001a). The uptake ofinert particles across the GI tract is known to occur mainly transcellularlythrough normal enterocytes and Peyer’s patches via M-cells (Hussain et al.,2001). Considering nanoparticulate vaccines for oral administration, theantigen is only released in the lymphoid tissue to induce the immuneresponse after Peyer’s patch internalization of the particles (Van DerLubben et al., 2001a). Therefore, uptake studies into Peyer’s patches haveextreme importance on the evaluation of the potential of the particles as anoral antigen delivery system. The uptake studies can be performed with ratswith a weight ranging between 250 and 350 g. On the day before theexperiment, animals are starved overnight, only with free access to water.

The rats are anesthetized by IM administration of 0.5 ml/kg of HypnormÒ

(fentanyl citrate 0.315 mg/ml and fluanisone 10 mg/ml) and 0.5 ml/kg ofDormicumÒ (midazolam 5 mg/lm). The animals need to remain anesthe-tized throughout the experiment and are placed on electrical heating mats.A small incision is made in the lower stomach and a Teflon tube (�: 0.5 mmI.D.�1.0 mmO.D.) is introduced through the pylorus approximately 3–5 cminto the duodenum. Fluorescent particles are placed (�500 ml) into theduodenum through the Teflon tube, and the incision is closed after theremoval of the tube from the stomach. The rats are sacrificed after 2 h bycervical dislocation. The whole intestine is removed and flushed with 20 ml ofcold (�4 �C) PBS. Between four and five Peyer’s patches can be excised fromeach intestine. They are fixed with 2% paraformaldehyde, and rinsed againwith PBS (4 �C), and the tissue is then permeabilized by immersion in 0.1%


Triton X-100 (in PBS) for 20 min. The tissue is rinsed again and stained with a0.0617% solution of BODIPYÒ 665/676 (Pierce) in methanol for 60 min.Finally, the Peyer’s patches are mounted on glass slides and observed using aconfocal laser scanning microscope.

9. Concluding Remarks

The development of novel vaccine adjuvants is becoming as importantas the development of novel antigens itself. Presently, most of the vaccinesare given by intramuscular injection, which requires the use of needlesthat are painful, are potentially dangerous, requires trained medical person-nel, and are therefore unsuitable for mass vaccination campaigns, especiallyin developing countries. Recently, many researchers have focused theirinterest on needle-free technologies for immunization, including a varietyof approaches for mucosal and topical immunization. Although severalstrategies have been proposed, some with very promising results, there isnot an approved needle-free vaccine against HBV so far, most likely becauseregulatory entities tend to adopt a cautious approach toward novel adjuvantsand administration routes in terms of safety in humans. The design ofchitosan-based antigen delivery systems has been explored by a considerablenumber of researchers mainly with the purpose of finding a good mucosaladjuvant. Therefore, it is prudent that a considerable number of in vitro testswould be performed to prove the efficacy of the delivery system as anadjuvant, before starting the immunization studies.

REFERENCES

Baldrick, P. (2010). The safety of chitosan as a pharmaceutical excipient. Regul. Toxicol.Pharmacol. 56, 290–299.

Berthold, A., Cremer, K., and Kreuter, J. (1996). Preparation and characterization ofchitosan microspheres as drug carrier for prednisolone sodium phosphate as model foranti-inflammatory drugs. J. Control. Release 39, 17–25.

Bessa, J., and Bachmann, M. F. (2010). T cell-dependent and -independent IgA responses:Role of TLR signalling. Immunol. Invest. 39, 407–428.

Bilensoy, E., Sarisozen, C., Esendagli, G., Dogan, A. L., Aktas, Y., Sen, M., andMungan, N. A. (2009). Intravesical cationic nanoparticles of chitosan and polycaprolac-tone for the delivery of Mitomycin C to bladder tumors. Int. J. Pharm. 371, 170–176.

Borges, O., Borchard, G., de Sousa, A., Junginger, H. E., and Cordeiro-da-Silva, A. (2007a).Induction of lymphocytes activated marker CD69 following exposure to chitosan andalginate biopolymers. Int. J. Pharm. 337, 254–264.

Borges, O., Borchard, G., Verhoef, J. C., de Sousa, A., and Junginger, H. E. (2005).Preparation of coated nanoparticles for a new mucosal vaccine delivery system. Int.J. Pharm. 299, 155–166.


Borges, O., Cordeiro-da-Silva, A., Romeijn, S. G., Amidi, M., de Sousa, A., Borchard, G.,and Junginger, H. E. (2006). Uptake studies in rat Peyer’s patches, cytotoxicityand release studies of alginate coated chitosan nanoparticles for mucosal vaccination.J. Control. Release 114, 348–358.

Borges, O., Tavares, J., de Sousa, A., Borchard, G., Junginger, H. E., and Cordeiro-da-Silva, A. (2007b). Evaluation of the immune response following a short oral vaccinationschedule with hepatitis B antigen encapsulated into alginate-coated chitosan nanoparti-cles. Eur. J. Pharm. Sci. 32, 278–290.

De, S., and Robinson, D. (2003). Polymer relationships during preparation of chitosan-alginate and poly-L-lysine-alginate nanospheres. J. Control. Release 89, 101–112.

Fessi, H., Devissaguet, J. P., Puisieux, F., and Theis, C. (1992). Process for the preparation ofdispersible colloidal systems of a substance in the form of nanoparticles. United StatesPatent 5118528.

Gallagher, S., and Smith, J. (2003). One-dimensional gel electrophoresis of proteins. Curr.Protoc. Immunol. 8.4, 1–21.

Gonzalez Ferreiro, M., Tillman, L., Hardee, G., and Bodmeier, R. (2002). Characterizationof alginate/poly-L-lysine particles as antisense oligonucleotide carriers. Int. J. Pharm. 239,47–59.

Hussain, N., Jaitley, V., and Florence, A. T. (2001). Recent advances in the understandingof uptake of microparticulates across the gastrointestinal lymphatics. Adv. Drug Deliv.Rev. 50, 107–142.

Illum, L. (1998). Chitosan and its use as a pharmaceutical excipient. Pharm. Res. 15,1326–1331.

Jabbal-Gill, I. (2010). Nasal vaccine innovation. J. Drug Target. 18, 771–786.Jung, T., Kamm, W., Breitenbach, A., Kaiserling, E., Xiao, J. X., and Kissel, T. (2000).

Biodegradable nanoparticles for oral delivery of peptides: Is there a role for polymers toaffect mucosal uptake? Eur. J. Pharm. Biopharm. 50, 147–160.

Kasturi, S. P., Skountzou, I., Albrecht, R. A., Koutsonanos, D., Hua, T., Nakaya, H. I.,Ravindran, R., Stewart, S., Alam, M., Kwissa, M., Villinger, F., Murthy, N., et al.(2011). Programming the magnitude and persistence of antibody responses with innateimmunity. Nature 470, 543–547.

Kim, B., Bowersock, T., Griebel, P., Kidane, A., Babiuk, L. A., Sanchez, M., Attah-Poku, S., Kaushik, R. S., and Mutwiri, G. K. (2002). Mucosal immune responsesfollowing oral immunization with rotavirus antigens encapsulated in alginate micro-spheres. J. Control. Release 85, 191–202.

Lebre, F., Borchard, G., de Lima, M. C., and Borges, O. (2011). Progress towards a needle-free hepatitis B vaccine. Pharm. Res. 28(5), 986–1012.

Malvern. (2004). Zetasizer Nano Series. User Manual. MAN 031, Issue 2.1.Oyewumi, M. O., Kumar, A., and Cui, Z. (2010). Nano-microparticles as immune adju-

vants: Correlating particle sizes and the resultant immune responses. Expert Rev. Vaccines9, 1095–1107.

Panos, I., Acosta, N., and Heras, A. (2008). New drug delivery systems based on chitosan.Curr. Drug Discov. Technol. 5, 333–341.

Pecora, R. (2000). Dynamic light scattering measurement of nanometer particles in liquids.J. Nanopart. Res. 2, 123–131.

Rajaonarivony, M., Vauthier, C., Couarraze, G., Puisieux, F., and Couvreur, P. (1993).Development of a new drug carrier made from alginate. J. Pharm. Sci. 82, 912–917.

Roy, K., Mao, H. Q., Huang, S. K., and Leong, K. W. (1999). Oral gene delivery withchitosan–DNA nanoparticles generates immunologic protection in a murine model ofpeanut allergy. Nat. Med. 5, 387–391.

Thanavala, Y., Lavelle, E., and Ogra, P. (2009). All things mucosal. Expert Rev. Vaccines 8,139–142.


Van Der Lubben, I. M., Konings, F. A., Borchard, G., Verhoef, J. C., and Junginger, H. E.(2001a). In vivo uptake of chitosan microparticles by murine Peyer’s patches: Visualiza-tion studies using confocal laser scanning microscopy and immunohistochemistry. J. DrugTarget. 9, 39–47.

van der Lubben, I. M., Verhoef, J. C., Borchard, G., and Junginger, H. E. (2001b). Chitosanfor mucosal vaccination. Adv. Drug Deliv. Rev. 52, 139–144.

Wee, S., and Gombotz, W. R. (1998). Protein release from alginate matrices. Adv. DrugDeliv. Rev. 31, 267–285.

Documents

[Methods in Enzymology] Nanomedicine - Infectious Diseases, Immunotherapy, Diagnostics, Antifibrotics, Toxicology and Gene Medicine Volume 509 || Chitosan-Based Nanoparticles as a