M-cell targeted biodegradable PLGA nanoparticles for oralimmunization against hepatitis B

PREM N. GUPTA, KAPIL KHATRI, AMIT K. GOYAL, NEERAJ MISHRA, & SURESH P. VYAS

Drug Delivery Research Laboratory, Department of Pharmaceutical Sciences, Dr Harisingh Gour Vishwavidyalaya, Sagar

470003, MP, India

(Received 22 May 2007; revised 11 August 2007; accepted 18 August 2007)

AbstractThe transcytotic capability and expression of distinct carbohydrate receptors on the intestinal M-cells render it a potentialportal for the targeted oral vaccine delivery. PLGA nanoparticles loaded with HBsAg were developed and antigen wasstabilized by co-encapsulation of trehalose and Mg(OH)2. Additionally, Ulex europaeus 1 (UEA-1) lectin was anchored to thenanoparticles to target them to M-cells of the peye’s patches. The developed systems was characterized for shape, size,polydispersity index and loading efficiency. Bovine submaxillary mucin (BSM) was used as a biological model for the in vitrodetermination of lectin activity and specificity. The targeting potential of the lectinized nanoparticles were determined byConfocal Laser Scanning Microscopy (CLSM) using dual staining technique. The immune stimulating potential wasdetermined by measuring the anti-HBsAg titre in the serum of Balb/c mice orally immunized with various lectinizedformulations and immune response was compared with the alum-HBsAg given intramuscularly. Induction of the mucosalimmunity was assessed by estimating secretary IgA (sIgA) level in the salivary, intestinal and vaginal secretion. Additionally,cytokines (interleukin-2; IL-2 and interferon-g; IFN-g) level in the spleen homogenates was also determined. The resultssuggest that HBsAg can be successfully stabilized by co-encapsulation of protein stabilizers. The lectinized nanoparticles havedemonstrated approximately 4-fold increase in the degree of interaction with the BSM as compared to plain nanoparticles andsugar specificity of the lectinized nanoparticles was also maintained. CLSM showed that lectinized nanoparticles werepredominantly associated to M-cells. The serum anti-HBsAg titre obtained after oral immunization with HBsAg loadedstabilized lectinized nanoparticles was comparable with the titre recorded after alum-HBsAg given intramuscularly. Thestabilized UEA-1 coupled nanopartilces exhibited enhanced immune response as compared to stabilized non-lectinizednanoparticles. Furthermore, the stabilized lectinized nanoparticles elicited sIgA in the mucosal secretion and IL-2 and IFN-gin the spleen homogenates. These stabilized lectinized nanoparticles could be a promising carrier-adjuvant for the targetedoral-mucosal immunization.

Keywords: Oral immunization, HBsAg, Ulex europaeus 1, PLGA, vaccine delivery

Introduction

The oral vaccine delivery is fascinating for many

reasons including the ease of delivery, compliance and

potential to induce mucosal immunity. Since a vast

majority of the human diseases are transmitted via the

mucosae, the induction of the protective immunity at

these sites could provide a highly effective means to

prevent infections (Berzofsky et al. 2001). Unfortu-

nately, harsh gastrointestinal conditions and poor

immunogenicity of many purified antigen generally

render oral vaccine delivery ineffective (Lavelle et al.

2004). Various strategies and delivery systems have

been devised for effective oral vaccine delivery.

Microparticles/nanoparticles delivery systems are

particularly useful to protect the antigen in the

gastrointestinal tract and hold potential to enhance

the efficacy of oral vaccine (O’Hagan 1998).

Polymerized liposomes, which are having improved

stability in gastrointestinal tract, have also been

ISSN 1061-186X print/ISSN 1029-2330 online q 2007 Informa UK Ltd.

DOI: 10.1080/10611860701637982

Correspondence: S. P. Vyas, Drug Delivery Research Laboratory, Department of Pharmaceutical Sciences, Dr Harisingh GourVishwavidyalaya, Sagar 470003, MP, India. Tel: 91 758 226 5525. Fax: 91 758 222 65525. E-mail: vyas_sp@rediffmail.com

Journal of Drug Targeting, December 2007; 15(10): 701–713

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exploited for oral immunization (Chen et al. 1996).

Enhancing the specific binding of particulates to

intestinal mucosa using some cell selective ligands and

subsequent translocation/uptake by cells of gastroin-

testinal lining is another approach for the effective oral

vaccine delivery (Lavelle et al. 2001).

Among the various strategies to enhance the

efficiency of the oral vaccines the targeting of the

vaccines to the gateway of immune system; M-cells, is

envisages as a potential approach. M-cells are

characterized by their poorly organized brush border,

high endocytic activity and basolateral lymphocyte-

containing pocket (Sansonetti and Phalipon 1999).

They comprise nearly 5% of the cells within human

follicle associated epithelium and are involved in

antigen sampling for presentation to organized

lymphoid tissue beneath (Jepson and Clark 1998).

M-cells transcytose the antigen from the gut lumen to

the underlying lymphoid tissues, thereby permitting

the generation of a mucosal immune response.

Consequently M-cells may represent an efficient

portal for the oral vaccine delivery. A distinctive

glycoconjugate profile in the M-cell glycocalyx of mice

and other species could play a role in the specific

targeting of the microorganism to the uptake site of the

Peyer’s patches. In the near future, it may be possible

to exploit these surface glycoconjugate for the

targeting orally administered delivery system based

vaccines or drug to the specialized site of uptake.

During last two decades biodegradable poly (D,L-

lactic-co-glycolic acid) (PLGA) based microparticles/-

nanoparticles have been explored extensively

(Gutierro et al. 2002; Yeh et al. 2002; Katz et al.

2003). Binding and uptake of the particles was

enhanced when particles were conjugated to B subunit

of E. coli heat labile enterotoxin (LTB), the plant

lectin, ConA or vitamin B12 following oral delivery to

rats (Russell-Jones 2001). Covalent attachment of

Ulex europaceous agglutinin 1 (UEA-1) to polystyrene

microspheres and oral delivery to mice result in

selective binding to and rapid uptake by the Peyer’s

patch M-cells (Foster et al. 1998). Orally adminis-

tered polystyrene microparticles with attached Lyco-

persicum esculentum agglutinin (LEA) were taken to a

greater extent than unconjugated particles in the rats

(Florence et al. 1995). The linkage of sepharose beads

to WGA and solanum tuberosum lectin (STL)

enhanced their binding to caco-2 cells (Gabor et al.

1995). These observations and a body of additional

data suggest that lectins are potential tools for the

enhanced binding and internalization of orally

delivered drugs and drug delivery systems and

efficiency of oral vaccines can be improved by M-cell

targeting.

The incorporation of proteins in the PLGA

microparticles/nanoparticles suffers significant pro-

tein degradation during preparation, storage and

following in vivo administration. Among the various

strategies to prevent interface-induced protein dena-

turation and aggregation, the addition of polyol or

sugar excipients in the aqueous phase is well

documented (Cleland and Jones 1996; Perez and

Griebenow 2001). Further, acidity commonly devel-

ops in PLGA formulations because of accumulation of

acidic degradation products upon polyester hydrolysis

(Shenderova et al. 1999) and the acidic microenvir-

onment of PLGA delivery systems is a potential cause

of instability of encapsulated proteins. Co-encapsula-

tion of protein stabilizer, trehalose and Mg(OH)2 has

been proved as an potential approach in our previous

investigations for the antigen stabilization within

PLGA carrier constructs (Jaganathan et al. 2004;

Gupta et al. 2006).

Proteins with lectin or lectin-like properties are

effective mucosal immunogen and there is a

relationship between receptor binding in the gut and

mucosal immunogenicity (De Aizpurua and Russell-

Jones 1998). However, despite a number of studies on

the lectin binding and evidence that plant lectins

conjugated to antigen/hapten may enhance immune

response following oral or intranasal delivery, the data

are trivial on the lectinized delivery systems and their

potential in the elicitation of the immune response to

encapsulated antigen. Mice M-cells express a-L-

fucose residue on their apical surface and UEA-1 has

a-L-fucose specificity (Foster et al. 1998). Thus, the

present study was undertaken for the development of

M-cell targeted UEA1 anchored HBsAg loaded

nanoparticles for targeted oral-mucosal vaccine

delivery. The HBsAg loaded PLGA nanoparticles

were prepared and antigen was stabilized in the

nanoparticles by the co-encapsulation of trehalose and

Mg(OH)2. UEA-1 was anchored on the nanoparticles

to confer on them M-cell targeting potential.

Lectinized nanoparticles were evaluated for various

in vitro parameters and they were assessed in mice for

their capability to induce systemic and mucosal

immune response. Additionally cytokine levels (IL-2

and IFN-g) have also been determined.

Materials and method

Materials

PLGA with a lactide to glycolide ratio of 50:50 (MW

40,000–75,000 Da), polyvinyl alcohol (MW 30,000–

70,000 Da), Ulex europaeus 1(UEA 1), FITC-UEA1,

TRITC-UEA1, FITC-BSA and CHAPS were pro-

cured from Sigma Chemical Co. (St Louis, MO,

USA). Glutaraldehyde (25% in water) was purchased

from Fluka Chemica Co. (AG CH-9470 Buchs,

Switzerland). HBsAg (MW 24 kDa, 1.5 mg/ml) was

obtained from Panacea biotech Ltd. (Lalru, Punjab,

India). Enzyme linked immunoassay kit (AUSAB and

AUZYME) and cytokines (IL-2 and IFN-g) esti-

mation kit was purchased from Abbott Laboratories,

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USA and e-Bioscience respectively. All other chemi-

cals and reagents were of analytical grade.

Preparation of PLGA nanoparticles

PLGA nanoparticles were prepared by double emul-

sion method as we have reported previously (Gupta

et al. 2006) with slight modifications. Briefly, to the

1 ml aqueous phase 600ml of recombinant HBsAg,

1.5% w/v trehalose and 2% w/v Mg(OH)2 was added

which was further suspended in 10 ml of 4% w/v PLGA

in dichloromethane. The mixture was probe sonicated

(Soniweld, India) for 2 min at 40 W in an ice bath.

To this water-in-oil emulsion, 40 ml of 5% w/v aqueous

polyvinyl alcohol was added and probe sonicated for

3 min to obtain a w/o/w emulsion. The emulsion was

stirred vigorously for 3 h. The nanoparticles were

collected by centrifugation, washed twice with distilled

water to remove PVA and then lyophilized.

Preparation of UEA-1 coupled PLGA nanoparticle

UEA1 was covalently coupled to PVA associated to

the surface of nanoparticles by method as reported

previously (Montisci et al. 2001) with slight modifi-

cation. The method involves two-steps; activation of

hydroxyl group of surface associated PVA followed by

its coupling with lectin.

Activation of hydroxyl group of nanoparticle by using

glutaraldehyde. Fifty milligrams of the nanoparticles

were washed in milli-Q water by centrifugation

(20,000g, 15 min). The pellet was resuspended by

vortexing in 750ml of milli-Q water and 1 ml of

glutaraldehyde (25% aqueous solution) and 250ml of

0.3 M H2SO4 were added. The mixture was then

shaked gently for 1 h at 308C to activate hydroxyl

group of surface-anchored PVA.

Conjugation of nanoparticles with lectins. The unreacted

glutaraldehyde was removed by centrifugation. Any

remaining traces of glutaraldehyde were removed by

three washing in phosphate buffer saline (PBS

10 mM, pH 7.4). Then 900ml of PBS containing

250mg of UEA1 was added for surfacial conjugation

by incubation overnight at room temperature. The

conjugates were centrifuged to remove free lectins and

incubated 1 h with ethanolamine (0.1 M) to mask

unreacted groups on the particles. The ethanolamine

was removed and nanoparticles were washed three

times by centrifugation. The lectin-coupled

nanoparticles were finally resuspended in 1 ml PBS

and stored at 48C.

Determination of the amount of bound lectin

The amount of UEA1 coupled to nanoparticles was

determined as the difference between the lectin added

initially and the lectin recovered in the solution after

incubation with the particles (Montisci et al. 2001).

The amount of lectin was quantified by the colori-

metric determination of protein in the supernatant by

bicinchoninic protein assay (BCA protein kit, Genie,

Bangalore).

Stability of surface modified nanoparticles

The method reported by Ertl et al. (2000) was used to

investigate the stability of conjugation between lectin

and PLGA nanoparticles. FITC-UEA1 was used for

the surface anchoring with PLGA nanoparticles. Ten

milligram of FITC-UEA1 coupled nanoparticles were

mixed with 1 ml of HEPES buffer at pH 7.4 at

48C.The supernatant was analyzed at regular intervals

by using spectrofluorimeter (SPECTRA max

GEMINI XPS, Molecular Device) after centrifu-

gation at 22,000g for 15 min. at 48C. The aliquot

removed was replaced by fresh HEPES buffer.

Morphology and particle size analysis

The nanoparticles were observed for their surface

morphology by scanning electron microscopy (SEM,

JEOL 6100, Japan). The nanoparticles were placed on

the sample holders, sputter coated with gold and then

placed in SEM. The mean diameter and polydisper-

sity index of the nanoparticles was determined by

Zetasizer (Nano-ZS90, UK).

Protein loading efficiency

The loading efficiency of the HBsAg in the plain

PLGA nanoparticles and lectinized nanoparticles was

determined by dissolving 5 mg the nanoparticles in the

2 ml of 5% w/v sodium dodecyl sulphate in 0.1 M

sodium hydroxide solution (Singh et al. 1997). The

amount of the antigen was determined by AUZYME

monoclonal kit (Abbott Laboratories, Abbott Park,

IL, USA).

In vitro release of HBsAg

The in vitro release of HBsAg from PLGA nanopar-

ticles was carried out in PBS (pH 7.4). Vials

containing 40 mg of nanoparticles and 5 ml of PBS

(pH 7.4) were incubated at 378C on a constant

shaking mixer. At appropriate intervals 1.0 ml of

release medium was collected following centrifugation

at 22,000g for 20 min and 1.0 ml of fresh PBS (pH

7.4) was again added to the vial. The amount of

HBsAg released was estimated by AUZYME mono-

clonal kit (Abbott Laboratories). The same sample

M-cell targeted nanoparticles 703

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was used to measure in vitro antigenicity using an

enzyme immunoassay (EIA) kit (AUSZYME; Abbott

Laboratories) as described by Shi et al. (2002). The

in vitro antigenicity of HBsAg was evaluated by using

the ratio of the EIA response to protein concentration

(EIA/protein).

In vitro ligand affinity and activity studies

The activity of the nanoparticles coupled with UEA-1

towards exogenously provided bovine submaxillary

gland mucin (BSM) and affinity toward competing

sugar were studied to assess the targeting efficacy of

ligand-anchored nanoparticles (Ezpeleta et al. 1996).

The in vitro targeting potential was determined by

mixing 1 ml of BSM in PBS (0.5 mg/ml) and same

volume of suspension of UEA-1 coupled nanoparti-

cles in PBS. After 60 min samples were centrifuged at

22,000g for 20 min, the aliquots of the supernatant

were taken and 20ml was injected into the HPLC

system (Ezpeleta et al. 1996). The amount of

interacted BSM was calculated as difference between

the total and the remaining BSM in the clear

supernatant. To study specificity, specific sugar (a-L-

fucose; 100 mM) and non-specific sugar (D-galactose;

100 mM) was added separately to the BSM bulk

solution in PBS and interaction of UEA-1 coupled

nanoparticles and BSM was determined.

M-cell targeting study

Dual staining (Clark et al. 1993) was used to assess

targeting potential of the developed delivery system.

Approximately 1 cm length of small intestine contain-

ing Peyer’s patch were excised, opened longitudinally

and pinned flat on corkboard. Tissue were rinsed

thoroughly with PBS (pH 7.4) and then cut in small

pieces (approximately 1 mm thick). The tissues were

subjected to dual staining with two lectin, by

immersion for 60 min in TRITC-UEA1, rinsing in

PBS and immersion for a further 60 min in FITC-

UEA1 coupled nanoparticles. Microtomy of the tissue

was performed using standard protocols and the thin

sections were viewed under confocal laser scanning

laser microscope (Bio-Rad, MRC 1024, UK).

Ex vivo specificity of lectin binding

The specificity of the lectin-coupled nanoparticles

towards receptors at the M-cell Peyer’s patches was

determined by method as reported previously with

slight modifications (Clark et al. 1993). The FITC-

UEA1 conjugated nanoparticles were first incubated

for 60 min at room temperature in PBS containing

carbohydrate inhibitor (a-L-fucose, 100 mM). The

Peyer’s patch tissue were then immersed in the

solution for 60 min at RT, rinsed in PBS and mounted

and examined by CLSM.

Immunization protocols

Animals and inoculations. Female BALB/c mice aged

8–10 weeks were used for in vivo studies. Animals

were housed in groups of six with free access to food

and water. They were deprived of food overnight prior

to oral immunization. The studies were carried out as

per the guidelines of Council for the Purpose of

Control and Supervision of Experiments on Animals

(CPCSEA), Ministry of Social Justice and

Empowerment, Government of India and the study

protocols was approved by Institutional Animals

Ethical Committee of Dr Hari Singh Gour

University, Sagar (MP), INDIA. The mice were

immunized orally with preparations equivalent to

10mg of HBsAg by three primary inoculations for 3

consecutive days. Booster immunization was done

after 3 weeks. Single intramuscular immunization with

booster dose after 3 weeks was also carried out with

alum-HBsAg to serve as standard. Various

formulation used for the immunization is shown in

Table I.

Collection of fluid. Subsequent to immunization blood

was collected after 2, 4, 6 and 8 weeks. Serum was

obtained by centrifugation of blood samples collected

from retro-orbital plexus of mice under ether

anesthesia and sera was stored at 2408C until

estimated for the antibody level by ELISA. The

salivary, intestinal and vaginal secretions were

collected after 5 weeks of booster immunization. For

collection of saliva, mice were injected 0.2 ml sterile

solution of pilocarpine (10 mg/ml) intraperitoneally

(IP). The mice began to salivate after approximately

2 min and the saliva was collected by using capillary

tube. Intestinal lavage was performed using the

technique as reported previously (Elson et al. 1984).

Briefly, four doses of 0.5 ml lavage solution (NaCl

25 mM, Na2SO4 40 mM, KCl 10 mM, NaHCO3

Table I. HBsAg loaded vaccine formulation for immunization

study.

Formulation code Description

HB-IM Intramuscularly given alum-HBsAg

HB-oral Orally given alum-HBsAg

HB-NP HBsAg loaded PLGA nanoparticles

HB-NP-UEA1 HBsAg loaded UEA 1

anchored PLGA nanoparticles

HB-NP-UEA1-T HBsAg loaded UEA1 anchored

PLGA nanoparticles stabilized with

trehalose

HB-NP-UEA1-T-M HBsAg loaded UEA1 anchored

PLGA nanoparticles stabilized with

trehalose and Mg(OH)2

HB-NP-T-M HBsAg loaded non-lectinized nanoparticles

stabilized with trehalose and

Mg(OH)2

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10 mM and polyethylene glycol-MW 3350; 48.5 mM)

were administered intragastrically at 15 min intervals

using a blunt tipped feeding needle. Thirty minutes

after the last dose of lavage solution the mice were

given 0.2 ml pilocarpine (10 mg/ml) IP. A discharge of

intestinal contents occurs regularly over next 20 min,

which was collected carefully. Vaginal secretions were

collected by using a pipettor to douche the mice with

0.1 ml of PBS (pH 7.4), which was then aspirated back

into the pipette tip and used for determination of

antibody levels. In order to increase the volume of

fluid available for assay, wherever necessary, samples

from two mice of identical immunized groups were

pooled. These fluids were stored with 100 mM

phenylmethyl sulfonyl fluoride (PMSF) as a protease

inhibitor at 2408C until tested by ELISA for secretory

antibody (sIgA) levels. Another group of animals were

sacrificed after 5 weeks of booster immunization and

spleens were removed for the determination of

endogenous cytokines levels (interferon-g and

interleukin-2).

Measurement of specific anti-HBsAg antibody

The concentration of anti-HBsAg antibody in the

collected serum sample was determined by using

commercially available solid-phase enzyme-linked

immunoassay kit (AUSABw, Abbott Laboratories).

Antibodies present in sera were estimated using 1/100

dilution as the first dilution of the serum. To signify

actual antibody concentration (antibody titre) in

mIU/ml, a standard curve was prepared using the

calibrated anti-hepatitis B panel provided by Abbott

Laboratories. Antibody response was plotted as log of

anti-HBsAg antibody titres (mIU/ml) versus time in

days.

Determination of IgA

Secretory IgA level in mucosal fluids and serum was

determined by ELISA method (Elson et al. 1984) with

slight modifications. Briefly, microtiter plates (Nunc-

Immune Platew Fb96 Maxisorb, Nunc, India) were

coated with a solution of HBsAg at 2mg/ml in

carbonate buffer (pH 9.6) for overnight at 48C. Wells

were blocked with PBS–BSA (3% (w/v)) for 1 h. The

plates were washed three times with 300ml of PBS

containing 0.05% Tween 20. Serial dilutions of

mucosal fluid in PBS–BSA (0.1% (w/v)) were added

and the plates were held at room temperature for 2 h

followed by washing and addition of horseradish

peroxidase-conjugated goat anti-mouse IgA (Sigma,

USA). IgA antibodies present in mucosal samples

were analyzed using 1/10 dilution as the first dilution

of the sample. After 1 h incubation and washing,

100ml of o-phenylenediamine dichloride (OPD;

Sigma, USA) in phosphate-citrate buffer (pH 5.5)

and H2O2 was added as a substrate. Colour

development was stopped after 30 min via the addition

of 50ml of 1N H2SO4 and the absorbance was

measured at 490 nm. The end point titre was

expressed as the logarithm of the reciprocal of the

last dilution, which gave an optical density at 490 nm

above the optical density of negative control.

Estimation of cytokine levels

Endogenous levels of IL-2 and IFN-g in mouse spleen

homogenates were estimated by using separate ELISA

kits for these cytokines (e-Biosciences) according to

the manual instructions. Spleen homogenates were

prepared by method reported by Nakane et al. (1992)

with slight modifications. Briefly, spleens were

weighed and homogenized in ice-cold PBS containing

1% CHAPS (Sigma) and 10% (w/v) homogenates

were obtained with the help of tissue homogenizer

(York, New Delhi, India). Homogenates were incu-

bated in an ice-bath for 1–2 h at temperature below

08C and the insoluble matters were settled down.

Supernatant were centrifuged at 2000g for 20 min and

the clear supernatants were used for cytokines

estimation by selected ELISA method.

Statistical analysis

The results were presented as mean ^ standard

deviation. Statistical analysis was carried out using

Student’s t-test and statistical significance was

designated as p , 0.05.

Results and discussion

Characterization of plain PLGA nanoparticles

and lectinized PLGA nanoparticles

The PLGA nanoparticles were prepared by

double emulsion method. The loading efficiency of

Table II. Characteristics of plain nanoparticles and lectinized PLGA nanoparticles.

Parameters Plain nanoparticles Lectinized nanoparticles

Average diameter (nm) 380.32 425.47

Polydispersity Index 0.174 0.162

Antigen loading (%) 48.41 ^ 4.32 45.35 ^ 4.12

Amount of lectin bound (mg/mg) – 15.72 ^ 1.14

Coupling efficiency (%) – 20.32 ^ 1.15

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HBsAg-PLGA nanoparticles was 48.41 ^ 4.32% and

average particle size was measured to be 380.32 nm.

Characteristics of plain and lectinized nanoparticles

were compared in Table II. In the present investi-

gation, hydroxyl group of the PVA at the surface of the

nanoparticles were used for the conjugation of lectins.

A two-step procedure (Montisci et al. 2001) was

adopted for the conjugation of lectins to the

nanoparticles; first step involves the activation of

surface associated hydroxyl groups of the PVA of

nanoparticles while in the second step glutaraldehyde

was used to conjugate lectin to the activated hydroxyl

group of nanoparticles (Figure 1).

The surface morphology of HBsAg loaded PLGA

nanoparticles was investigated by using SEM. As

shown in Figure 2, no major differences could be

detected between plain and lectin-coupled nanopar-

ticles. Protein entrapment and anchoring with the

lectin do not affect the spherical shape and surface

visible texture of nanoparticles. Upon grafting of UEA

1 to the nanoparticles, the mean diameter of the

particles however increased marginally (Table II).

Figure 1. Schematic presentation of anchoring of lectin to surface hydroxyl group of PVA via glutaraldehyde.

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This may be attributed to the immobilization of the

lectin on the surface of the nanoparticles. The

percentage antigen loading of lectinized nanoparticles

was measured to be 45.35 ^ 4.12%. The percentage

antigen loading was recorded to be slightly low in the

case of lectinized nanoparticles. The decrease could

be attributed to the release of antigen from

nanoparticles on incubation employed for anchoring

of lectin to the surface of the nanoparticles.

The amount of UEA-1 coupled to the nanoparticles

was estimated to be 15.72 ^ 1.14mg lectin/mg

nanoparticles, which amounts to a coupling efficiency

of 20.32 ^ 1.15%.

To investigate the stability of linkage between

PLGA nanoparticles and lectin, FITC-UEA1 con-

jugated nanoparticles were incubated in HEPES

buffer at 48C. During incubation period of 20 days,

9.2% of the total amount of lectin was delodged from

nanoparticles, on treatment of these nanoparticles

with 5 M urea, which is known to disrupt non-covalent

interactions, additional 7.7% of the FITC-UEA-1 was

released from the nanoparticles. Nevertheless,

approximately 83% of the lectin was estimated to be

retained/bound on PLGA-nanoparticles.

In vitro antigen release

In vitro release study was conducted with lectinized

and plain nanoparticles as well as with protein

stabilized nanoparticles. Proteins may unfold and

aggregate at the o/w interface therefore one straight-

forward strategy toward stabilization is to minimize

exposure to this interface. The addition of polyol or

sugar excipients in the aqueous phase is well

documented as a strategy to prevent interface-induced

protein denaturation and aggregation (Cleland and

Jones 1996; Perez and Griebenow 2001). In the

present investigation, an attempt has been made to

stabilize the protein during both, the encapsulation

process and the release of protein from the nanopar-

ticles by co-encapsulating a protein stabilizer, i.e.

trehalose. In this case, the protein stabilizer (trehalose)

could prevent the antigens from the organic solvent

exposure via preferential hydration of the surface.

Further, acidity commonly develops in PLGA

formulations due to accumulation of acidic degra-

dation products upon polyester hydrolysis (Shender-

ova et al. 1999). The acidic microclimate in PLGA

delivery systems is a potential source of instability of

encapsulated proteins. Peptide bond hydrolysis is

particularly fast at acidic pH (Zhu et al. 2000).

A rational approach to deter the pH alteration was by

inclusion of basic additive in the formulations. A basic

salt Mg(OH)2 was thus incorporated into the

nanoparticles to lend them retain the structure and

biological activity of encapsulated proteins.

The HBsAg release pattern from various PLGA

nanoparticles is shown in Figure 3. The release pattern

were noted to be typically biphasic with an initial burst

release attributed to the release of surface associated

protein, followed by a slower release phase which may

be accounted for entrapped protein slow diffusion into

the release medium (Coombes et al. 1998). The

trehalose being hydrophilic in nature dissolves rapidly

from the polymeric sheath leaving porous matrix and

as a result the formulations containing trehalose

showed increased release profile (45.7 ^ 4.1% cumu-

lative release in 35 days) in comparison to the

formulation without trehalose (HB-NP and HB-NP-

UEA1 showed 20.8 ^ 1.9% and 29.4 ^ 2.7% cumu-

lative release in 35 days respectively). The higher

release of lectinized (HB-NP-UEA1) as compared to

plain nanoparticles (HB-NP) may be due to the

hydrophilic characteristics of lectin, allowing easier

penetration of aqueous solution into the matrix

thereby dissolving the protein (Walter et al. 2004).

The in vitro antigenicity of the antigen was evaluated in

terms of EIA/protein ratio (Figure 4). The antigenicity

of the lectinized formulation with the stabilizer

(trehalose and Mg(OH)2) was found to be

0.96 ^ 0.093 after 35 days. Plain nanoparticles and

lectinized nanoparticles without stabilizer showed

significantly (p , 0.05) lower antigenicity. The results

Figure 2. Scanning electron photomicrograph of plain

nanoparticles (A) and lectin anchored nanoparticles (B).

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are consistent with our previous findings (Jaganathan

et al. 2004; Gupta et al. 2006).

In vitro ligand affinity and activity studies

The presence of numerous functional groups

(i.e. amino and carboxylic residues) renders protein

an excellent candidate for the preparation of con-

jugates, through attachment of ligand capable of

providing specificity to the surface of nanoparticles

such as lectins. In the present investigation BSM

(bovine submaxillary mucin), a glycoprotein, was used

as a biological model to determine the in vitro activity

and specificity of UEA1-coupled nanoparticles

towards sugar residue of glycoprotein. The carbo-

hydrate part of the BSM is composed of six sugars;

N-acetylgalactosamine, N-acetylglucosamine, galac-

tose, mannose, fucose and sialic acid (Honda and

Suzuki 1984; Vyas et al. 2001a). For the determi-

nation of in vitro activity of UEA1, experiment was

carried out in the absence of specific sugar and for

the determination of in vitro specificity of UEA1, the

experiment was carried out in the presence of the

specific sugar (a-L-fucose) and non-specific or

control sugar (D-galactose) for UEA1. In the absence

of a-L-fucose and in the presence of D-galactose,

Figure 3. In vitro cumulative release of HBsAg from UEA1 anchored PLGA nanoparticles (with and without stabilizer) and plain

nanoparticles (n ¼ 4).

Figure 4. In vitro antigenicity (response of EIA to protein concentration) of HBsAg in lectin-coupled PLGA nanoparticles (with and without

stabilizer) and plain nanoparticles during in vitro release study.

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the UEA1-coupled nanoparticles exhibited almost

four times higher interaction with BSM than

unmodified nanoparticles (Figure 5). In the presence

of a-L-fucose, the interaction between UEA1 coupled

nanoparticles and BSM was significantly reduced.

Plain nanoparticles revealed fairly comparable

( p , 0.05) results in absence and in presence of

specific sugar for UEA1. Thus results suggest that

lectinized nanoparticles retain activity and same sugar

specificity as the native lectin UEA1.

Targeting of nanoparticles to M-cells

Polyvinyl alcohol is the most commonly used

emulsifier in the fabrication of the PLGA based

microparticles or nanoparticles. Polymer may provide

a firm anchorage with the PVA on entanglement of

polymeric chains at the surface or sub-surface of the

matrix resulting into a core-shell structure (Boury et al.

1997). We have reported previously that a fraction of

PVA remains associated with nanoparticles despite of

several washing because PVA forms an interconnected

network with the PLGA at the interface (Gupta et al.

2006). Thus, it is inferred that PVA offers a strong

adsorbed layer over nanoparticles. However, nano-

particles with higher amount of PVA have relatively

lower cellular uptake despite smaller particle size

(Sahoo et al. 2002). The lower intracellular uptake of

nanoparticles with higher amount of residual PVA is

attributed to the higher hydrophilicity of the nano-

particle surface. In order to facilitate the uptake and to

target the nanoparticle to the M-cells of the Peyer’s

patch, the anchoring of UEA1 to the surface of PLGA

nanoparticles have been envisaged.

The confocal laser scanning microscopy (CLSM)

was used to assess targeting potential of the lectinized

PLGA nanoparticles. The targeting of UEA1 coupled

PLGA nanoparticles was confirmed by dual staining

of the Peyer’s patches M-cells. M-cells were first

stained with TRITC-UEA1 followed by adminis-

tration of FITC-UEA1 anchored nanoparticle. As

shown in Figure 6 there was an enhancement in the

binding of lectinized nanoparticles as compared to

control nanoparticles (coated with FITC-BSA) that

showed little or no binding to the M-cell in mice.

Lectins are proteins or glycoproteins capable of

specific recognition of and reversible binding to

carbohydrate determinants of complex glycoconju-

gates, without altering the covalent structure of any of

the recognized glycosyl ligands. They are efficient in

recognizing the complex oligosaccharide epitopes,

which are also present on the cell surface or could

be exogenous glycoconjugate ligands mimics of

endogenous carbohydrate epitopes (Vyas et al.

2001b). The binding of the lectin to the corresponding

receptor is specific in nature. The specificity of the

lectin binding was assessed by pre-incubation of the

lectin (UEA1) with its specific sugar (a-L-fucose).

When FITC-UEA1 anchored nanoparticles were

Figure 5. Binding of BSM to UEA-anchored PLGA nanoparticles

(NPs-UEA1) and plain nanoparticles (NPs) in suspension with and

without competing sugar (a-L-fucose) and with non-specific or

control sugar (D-galactose; n ¼ 4).

Figure 6. Confocal laser scanning microscopy images showing

targeting of the nanoparticles to the M-cells of the Peyer’s patches in

mice by dual staining. M-cells were primarily stained with TRITC-

UEA1 (red). FITC-UEA1 coupled PLGA nanoparticles stain

green. Control nanoparticles (FITC-BSA coated) showed little or

no binding to M-cells (A). Lectinized nanoparticles (shown by

arrow) were associated predominantly with M-cells (B).

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incubated with a-L-fucose (100 mM) prior to incu-

bation with Peyer’s patch tissue the staining intensity

greatly reduced (Figure 7). Thus, incubation of the

lectinized nanoparticles with the corresponding sugar

inhibitor abolished the targeting potential of the

particles to the M-cells.

Immunological response

Prior to the oral immunization with the lectinized

nanoparticles the mice were deprived of food

overnight because the carbohydrate present in

ingested food can complex with lectins and prevent

interaction with uptake site receptors. The level of

anti-HBsAg antibodies was determined for all

experimental groups after 2, 4, 6 and 8 weeks. The

serum anti-HBsAg antibody titre was determined by

three inoculations in consecutive days and boosting

after the third week with the same formulation

(Figure 8). The in vivo evaluation showed that

HBsAg loaded lectinized nanoparticles (HB-NP-

UEA1) produced higher anti-HBsAg titre as com-

pared to plain nanoparticles (HB-NP). Similarly, the

trehalose and Mg(OH)2 based stabilized lectinized

nanoparticles (HB-NP-UEA1-T-M) revealed signifi-

cantly higher ( p , 0.05) antibody titre as compared to

stabilized nontargeted formulation (HB-NP-T-M).

This may be attributed to the targeting of vaccine

loaded lectinized nanoparticles to the antigen uptake

site of the M-cell of the Peyer’s patches by virtue of

UEA1. In addition to the role of plant lectin as

targeting agents, some of these molecules are highly

immunostimulatory and may have potential mucosal

adjuvant action (Lavelle et al. 2001). The trehalose

and Mg (OH)2 stabilized nanoparticles showed higher

immune response owing to the protective effect of the

stabilizer on the integrity of antigen which may

otherwise affected adversely by the exposure to

organic solvent during manufacturing process. The

results are in accordance to our in vitro investigation

showing that antigenicity of HBsAg in lectin-coupled

nanoparticels without stabilizer was low owing to the

denaturation of the antigen (Gupta et al. 2006). The

stabilized nanoparticles could shield the antigen

during various stressful conditions of manufacturing

Figure 7. Confocal laser scanning microscopy images showing

specificity of the lectin-coupled nanoparticles towards carbohydrate

receptors of the M-cells of the Peyer’s patches. UEA1-PLGA

nanoparticles were predominantly associated with the M-cells (A).

Incubation of the UEA1-PLGA nanoparticles with the a-L-fucose

(100 mM) result in significant reduction in the targeting of the

nanoparticles to M-cells (B).

Figure 8. Serum anti-HBsAg profile of mice immunized with

different formulations by three primary inoculations for 3

consecutive days. Booster immunization was given after 3 weeks.

The antibody titers obtained following oral immunization with

lectinized nanoparticles were compared with the titre obtained with

single intramuscular administration of alum-HBsAg and boosting

after 3 weeks with the same formulation. Values are expressed as

mean ^ SD (n ¼ 4).

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and subsequently it also confers protection while

antigen release.

When mice were immunized with three primary

inoculations for 3 consecutive days and boosting after

the third week, the anti-HBsAg antibody titre was

found to be equivalent for stabilized lectinized

nanoparticles (HB-NP-UEA1-T-M) administered

orally and alum-HBsAg given intramuscularly. In all

experimental groups the antibody titre was found to

be enhanced significantly ( p , 0.05) after boosting on

third week. The induction of the immune response

with the nanoparticles based formulations is attrib-

uted to the facilitation of the uptake of the antigen by

the Peyer’s patches. Further anchoring of the lectin

may result in avid uptake of the nanoparticles through

the M-cell. The uptake of the lectinized particles from

the gut has been previously described in mice (Foster

et al. 1998) and receptor mediated binding of the

lectin to the mucosa was considered as an important

determinant of mucosal immunity.

Mucosal IgA plays an important role in protection

against enteropathogens and viruses both in human

and animal models (Marcotte and Lavoie 1998).

Secretory IgA (sIgA) is principle antibody isotype

produced in the intestine. Specific sIgA is pivotal in

providing the protection against intestinal bacteria and

viruses and hence is an essential requirement of

effective oral vaccines. Moreover, production of

HBsAg-specific mucosal IgA antibodies must be

important for protection from mucosally transferred

HBV (Isaka et al. 2001). The sIgA response detected

was negligible for the intramuscular route of

immunization (Figure 9), whereas mucosal route of

administration produces significantly higher sIgA

level. The immunization of mice with three primary

inoculations for 3 consecutive days and boosting

after 3 weeks with stabilized lectinized nanoparticles

(HB-NP-UEA1-T-M) induces significantly higher

sIgA level as compared to stabilized untargeted

nanoparticles (HB-NP-T-M). Additionally, sIgA titre

obtained with untargeted plain nanoparticles (HB-

NP) and untargeted stabilized nanoparticles (HB-NP-

T-M) was significantly higher when compared to

HBsAg given orally or by intramuscular route

( p , 0.05).

Endogenous cytokine levels (IL-2 and IFN-g) were

determined in spleen homogenate after 5 weeks of

booster immunization of different formulations

(Figures 10 and 11). The significant levels of both

IL-2 and IFN-g were measured in mice immunized

with various lectinized nanoparticles as compared to

those recorded for control and unmodified PLGA

nanoparticles ( p , 0.05). Both Th1 dependent

cytokines are evidenced for the cell-mediated immune

response elicited by lectinized nanoparticles. The

activation of the Th1 subset is associated with the

production of IFN-g, and IL-2 and the development

of the classical cell mediated immune response. This is

in accordance to the previous report demonstrating

the significant production of Th1-cytokine (IL-2 and

IFN-g) by the lectinized microparticles (Roth-Watler

et al. 2005). Further, it has been argued that lectin

renders the accumulation of the antigen at the desired

mucosal sites, which may result in the induction of the

specific Th1 antibody response. Thus M-cell directed

vaccine loaded particles are potentially useful for the

immunomodulation toward Th1 in an ongoing Th2

response.

Soluble protein antigens are processed via the

exogenous pathways and are presented on the surface

of antigen presenting cell in context of MHC class II

glycoproteins to selectively stimulate CD4þT-cells.

On contrary, CD8þ CTL usually recognizes endogen-

ous antigens that are presented on the cell surface in

association with MHC class I molecules (Brodsky and

Guagliardi 1991). But there are some exceptions to

this rule. Some covalent modification of the protein

antigen such as lipid conjugation facilitate their access

to the endogenous processing pathway and their

Figure 9. sIgA level in the intestinal, salivary and vaginal secretion

of mice immunized with various formulation after 5 weeks of booster

immunization. Mice were immunized by three primary inoculations

for 3 consecutive days and booster dose was given after 3 weeks. For

intramuscular immunization single dose alum HBsAg was given and

boosting was done after 3 weeks with the same formulation. Values

are expressed as mean ^ SD (n ¼ 4).

Figure 10. Interleukin-2 level in spleen homogenate of mice

immunized with various formulations after 5 weeks of booster

immunization. Values are expressed as mean ^ SD (n ¼ 4).

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stimulation of a CD8þ CTL response in vivo (Deres

et al. 1989). Very little is known about the cell biology

of this type of processing by APC. The generation of a

dominant Th1 cytokine profile is important to

facilitate eradication of HBV infection and thus, it

can be utilized for therapeutic immunization of HBV

chronic carriers. This is in agreement with our

previous reports dealing with induction of Th1

cytokine production through mucosal immunization

(Jaganathan and Vyas 2006). Moreover, the Th1

cytokine (IFN-g) formation was the most typical

phenomena by using the M-cells targeting strategy

(Roth-Watler et al. 2004).

Conclusion

In conclusion, the advantages of the developed

delivery system is protection of the vaccine during

gastrointestinal transit by polymeric nanoparticles and

antigen within the nanoparticles are effectively

stabilized by co-encapsulation of protein stabilizer.

Further an efficient delivery of antigen to the mucosal

immune induction site (M-cell of the mice) was

achieved by surface anchoring with plant lectin UEA1

and therefore a directed accumulation of the desired

antigen to the intestinal immune system. This is

reflected in the heightened immune response obtained

with targeted nanoparticles. M-cell targeted vaccine

loaded nanoparticles elicited significantly higher

immune response as compared to non-targeted

nanoparticles. The induction of systemic, mucosal

and moderate cellular immunity has been observed

with stabilized M-cell targeted nanoparticles, which is

vital for the effective management of the various

infectious diseases and particularly viral infection.

Acknowledgements

One of the authors (Prem N. Gupta) acknowledges All

India Council for Technical Education (AICTE),

New Delhi, for the award of National Doctoral

Fellowship (Grant: 1-10/FD/NDF-PG/H.S.Gour

(44)/ 2005–2006). We are also grateful to SAIF

(Sophisticated Analytical Instrumental Facility), All

India Institutes of Medical Sciences, New Delhi, for

the SEM and CLSM.

References

Berzofsky JA, Ahlers JD, Belyakov IM. 2001. Strategies for

designing and optimizing new generation vaccines. Nat Rev

Immunol 1:209–219.

Boury F, Marchais H, Benoit JP, Proust JE. 1997. Surface

characterization of poly (a-hydroxy acid) microspheres prepared

by solvent evaporation/extraction process. Biomaterials 18:

125–136.

Brodsky FM, Guagliardi L. 1991. The cell biology of antigen

processing and presentation. Annu Rev Immunol 9:707–744.

Chen H, Torchilin V, Langer R. 1996. Lectin bearing polymerized

liposomes as potential oral vaccine carriers. Pharm Res 13:

1378–1383.

Clark MA, Jepson MA, Simmons NL, Booth TA, Hirst BH. 1993.

Differential expression of lectin binding sites defines mouse

intestinal M-cells. J Histochem Cytochem 41:1679–1687.

Cleland JL, Jones AJS. 1996. Stable formulation of recombinant

human growth hormones and interferon gamma for micro-

encapsulation in biodegradable microspheres. Pharm Res 13:

1464–1475.

Coombes AGA, Yeh MK, Lavelle EC, Davis SS. 1998. The control

of protein release from poly (DL-lactide-co-glycolide) micro-

particles by variation of external aqueous phase surfactant in the

water in oil in water method. J Control Release 52:311–320.

De Aizpurua HJ, Russell-Jones GJ. 1998. Identification of the

classes of the proteins that provide an immune response upon

oral feeding. J Exp Med 167:440–451.

Deres K, Schild H, Wiesmuller K-H, Jung G, Rammensee H-G.

1989. In vivo priming of virus specific cytotoxic T lymphocytes

with synthetic lipopeptide vaccine. Nature (London) 342:

561–564.

Elson CO, Ealding W, Lefkowitz J. 1984. A lavage technique

allowing repeated measurement of IgA antibody in mouse

intestinal secretions. J Immunol Methods 67:101–108.

Ertl B, Heigl F, Wirth M, Gabor F. 2000. Lectin mediated

bioadhesion: Preparation, stability and caco-2 binding of wheat

germ agglutinin-functionalized poly (D,L-lactic-co-glycolic acid)-

microspheres. J Drug Target 8:173–184.

Ezpeleta I, Irache JM, Stainmesse S, Chabenat C, Gueguen J,

Orecchioni AM. 1996. Preparation of lectin-vicilin nanoparticles

conjugate using the carbodiimide technique. Int J Pharm 142:

227–233.

Florence AT, Hillery A, Hussain N, Jani PU. 1995. Factors affecting

the oral uptake and translocation of polystyrene nanoparticles:

Histological and analytical evidence. J Drug Target 3:65–70.

Foster N, Clark MA, Jepson MA, Hirst BH. 1998. Ulex europaeus 1

lectin targets microspheres to mouse Peyer’s patch M-cells

in vivo. Vaccine 16:536–541.

Gabor F, Stangl M, Wirth M. 1995. Lectin mediated bioadhesion:

Binding characteristics of plant lectins on the enterocyte like cell

lines Caco-2, HT-29 and HCT-8. J Control Release 55:

131–142.

Gupta PN, Mahor S, Rawat A, Khatri K, Goyal A, Vyas SP. 2006.

Lectin anchored stabilized biodegradable nanoparticles for oral

immunization: 1. Development and in vitro evaluation. Int J

Pharm 318:163–173.

Gutierro I, Hernandez RM, Igartua M, Gascon AR, Pedraz JL.

2002. Size dependent immune response after subcutaneous, oral

Figure 11. Interferon-g level in spleen homogenate of mice

immunized with various formulations after 5 weeks of booster

immunization. Values are expressed as mean ^ SD (n ¼ 4).

P. N. Gupta et al.712

Jour

nal o

f D

rug

Tar

getin

g D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsita

ets-

und

Lan

desb

iblio

thek

Due

ssel

dorf

on

12/1

7/13

For

pers

onal

use

onl

y.

and intranasal administration of BSA loaded microspheres.

Vaccine 21:67–77.

Honda S, Suzuki S. 1984. Common conditions for high

performance liquid chromatographic micro determination of

aldoses, hexosamines and sialic acid in glycoproteins. Anal

Biochem 142:167–174.

Isaka M, Yasuda Y, Mizokami M, Kozuka S, Taniguchi T, Matano

K, Maeyama J, Mizuno K, Morokuma K, Ohkuma K, Goto N,

Tochikubo K. 2001. Mucosal immunization against hepatitis B

virus by intranasal co-administration of recombinant hepatitis B

surface antigen and recombinant cholera toxin B subunit as an

adjuvant. Vaccine 19:1460–1466.

Jaganathan KS, Vyas SP. 2006. Strong systemic and mucosal

immune responses to surface-modified PLGA microspheres

containing recombinant hepatitis B antigen administered

intranasally. Vaccine 24:4201–4211.

Jaganathan KS, Singh P, Prabakaran D, Mishra V, Vyas SP. 2004.

Development of a single-dose stabilized poly (DL-lactic-co-

glycolic acid) microspheres-based vaccine against hepatitis B.

J Pharm Pharmacol 56:1243–1250.

Jepson MA, Clark MA. 1998. Studying M cells and their role in

infection. Trends Microbiol 6:359–365.

Katz DE, Delorimier AJ, Wolf MK, Hall ER, Cassels FJ, van

Hamont JE, Newcomer RL, Davachi MA, Taylor DN,

McQueen CE. 2003. Oral immunization of adult volunteers

with microencapsulated enterotoxigenic Escherichia coli (ETEC)

C56 antigen. Vaccine 21:341–346.

Lavelle EC, Grant G, Pusztai A, fuller U, O’Hagan DT. 2001.

Identification of plant lectin with mucosal adjuvant activity.

Immunology 102:77–86.

Lavelle EC, Grant G, Pfuller U, O’Hagan DT. 2004. Immunologi-

cal implication of the use of plant lectins for drug and vaccine

targeting to the gastrointestinal tract. J Drug Target 12:89–95.

Marcotte H, Lavoie MC. 1998. Oral microbial ecology and the role

of salivary immunoglobulin A. Microbiol Mol Biol Rev 62:

71–109.

Montisci MJ, Giovannnuci G, Douchene D, Ponchel G. 2001.

Covalent coupling of asparagus pea and tomato lectin to poly

(lactide) microspheres. Int J Pharm 215:153–161.

Nakane A, Numata A, Minagawa T. 1992. Endogenous tumor

necrosis factor, interleukin-6, and gamma interferon levels

during Listeria monocytogenes infection in mice. Infect Immun

60:523–528.

O’ Hagan DT. 1998. Microparticles and polymers for the mucosal

delivery of vaccines. Adv Drug Deliv Rev 34:305–320.

Perez C, Griebenow K. 2001. Improved activity and stability of

lysozyme at the water/CH2Cl2 interface: Enzyme unfolding and

aggregation and its prevention by phenols. J Pharm Pharmacol

53:1217–1226.

Roth-Watler F, Scholl I, Untersmayr E, Fuchs R, Boltz-Nitulescu G,

Weissenbock A, Scheiner O, Gabor F, Jenson-Jarolim E. 2004.

M-cell targeting with Alenuria auerantia lectin as a novel

approach for oral allergen immunotherapy. J Allergy Clin

Immunol 114:1362–1368.

Roth-Watler F, Scholl I, Untersmayr E, Ellinger A, Boltz-Nitulescu G,

Scheiner O, Gabor F, Jenson-Jarolim E. 2005. Mucosal targeting

of allergen-loaded microspheres by Alenuria auerantia lectin.

Vaccine 23:2703–2710.

Russell-Jones GJ. 2001. The potential of receptor mediated

endocytosis for oral drug delivery. Adv Drug Deliv Rev 1:59–73.

Sahoo SK, Panyam J, Prabha S, Labhasetwar V. 2002. Residual

polyvinyl alcohal associated with poly (D, L-lactide-co-glycolide)

nanoparticles affects their physical properties and cellular

uptake. J Control Rel 82:105–114.

Sansonetti PJ, Phalipon A. 1999. M cells as port of entry for

enteroinvasive pathogens: Mechanism of interaction, conse-

quences for the disease process. Semin Immunol 11:193–203.

Shenderova A, Burke T, Schwenderman SP. 1999. The acidic

microclimate in poly (lactide-co-glycolide) microspheres stabil-

izes camptothecins. Pharm Res 16:241–248.

Shi L, Caulfield MJ, Chern RT, Wilson RA, Sanyal G, Volkin DB.

2002. Pharmaceutical and immunological evaluation of single-

shot hepatitis B vaccine formulated with PLGA microspheres.

J Pharm Sci 91:1019–1035.

Singh M, Li X, McGhee JP, Zamb T, Koff W, Wang CY, O’Hagan

DT. 1997. Controlled release microparticles as a single dose

hepatitis B vaccine: Evaluation of immunogenicity in mice.

Vaccine 15:475–481.

Vyas SP, Sihorkar V, Dubey PK. 2001a. Preparation, characteriz-

ation and in vitro antimicrobial activity of metronidazole bearing

lectinized liposomes for intra-peridontal pocket delivery.

Pharmazie 56:554–560.

Vyas SP, Singh A, Sihorkar V. 2001b. Ligand–receptor-mediated

drug delivery: An emerging paradigm in cellular drug targeting.

Crit Rev Ther Drug Carrier Syst 18:1–76.

Walter F, Scholl I, Untersmayr E, Ellinger A, Boltz-nitulescu G,

Scheiner O, Gabor F, Jensen-Jarolim E. 2004. Functionalization

of allergen loaded microspheres with wheat germ agglutinin for

targeting enterocytes. Biochem Biophys Res Comm 315:

281–287.

Yeh MK, Liu YT, Chen JL, Chiang CH. 2002. Oral immunogeni-

city of the inactivated Vibrio cholerae whole cell vaccine

encapsulated in biodegradable microparticles. J Control Release

82:237–247.

Zhu G, Millery SR, Schwendeman SP. 2000. Stabilization of protein

encapsulated in injectable poly (lactide-co-glycolide). Nat

Biotech 18:52–57.

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