Micro-organism-like nanoparticles for oral antigen delivery

J. DRUG DEL. SCI. TECH., 18 (1) 31-39 2008

Micro-organism-like nanoparticles for oral antigen delivery H.H. Salman1, S. Gómez1, C. Gamazo2, R. Costa Martins1, V. Zabaleta1, J.M. Irache1* Departamento de Farmacia y Tecnología Farmacéutica, Centro Galénico, University of Navarra, Apartado. 177, 31080 Pamplona, Spain 2 Departamento de Microbiologia, University of Navarra, Apartado. 177, 31080 Pamplona, Spain *Correspondence: [email protected] 1

The aim of this work was to design and evaluate microorganism-like nanoparticles able to either mimic adhesion and colonization patterns of bacteria or to enhance and modulate the host immune response. Thus poly(anhydride) nanoparticles were either coated with adhesive factors (either with flagellin from Salmonella enteritidis or mannosamine) or loaded with lipopolysaccharide of Brucella ovis. The mucosal affinity of the nanoparticles was investigated by bioadhesion and fluorescence microscopy studies. The immunoadjuvant properties were checked by the oral route using ovalbumin as an antigen model. The results indicated that both flagellin and mannosamine nanoparticles display a stronger mucosal affinity to normal mucosa and Peyer’s patches than control ones, whereas LPS entrapped ones did not. Surprisingly, LPS formulation was unable to enhance the immune response by the oral route. However, flagellin and mannosamine nanoparticles induced stronger and more balanced serum levels of both IgG1 (Th2) and IgG2a (Th1) than control ones. In addition, oral immunization with ligand formulations induced high levels of fecal IgA. Key words: Immunoadjuvant – Nanoparticles – Mannosamine – Flagellin – Lipopolysaccharide – Bioadhesion.

Until recently, most vaccines consisted of the entire micro-organism, alive or inactivated (bacterins), or attenuated toxins. Although all of these approaches have proved successful, several drawbacks related to their safety and security (residual virulence, undesirable side effects) and, in some cases, to the relatively poor efficacy against more challenging diseases (AIDS, malaria, tuberculosis) have limited their use. In the past decade, several new approaches to vaccine development have emerged that may have significant advantages over traditional ones. These new vaccines are based on the use of well-defined proteins, peptides or plasmid DNA. Although these “subunit vaccines” offer advantages such as reduced toxicity, they are poorly immunogenic when administered alone. For these reasons, technology is required to make these defined antigens immunogenic, including new strategies for the optimum physical presentation of the antigen [1, 2]. This is particularly challenging for the oral delivery of antigens. Mucosal immunization regimes that employ the oral route of delivery are often compromised by antigen degradation in the stomach as a result of the acidity, an extensive range of digestive enzymes in the intestine and a protective coating of mucus that limits access to the mucosal epithelium. Moreover, tolerance or immunological unresponsiveness to orally delivered vaccine antigens is also a major problem associated with this route of immunization. On the other hand, although its commercial viability is yet to be proven, oral vaccination provides a cost-effective and convenient alternative to conventional vaccines. In addition, an oral vaccine not only elicits a good systemic immune response but may also induce protection at the sites where pathogens often establish the infection. In the last years, efforts have been directed towards the enhancement of oral vaccine delivery. Most work has focused on the use of controlled-release polymeric nanoparticles as adjuvants for mucosal immunization [3-7]. In fact, antigens loaded in nanoparticles can be effectively protected from physicochemical and enzymatic degradation within the gastro-intestinal tract [8, 9]. In addition, nanoparticles can enhance the delivery of the loaded antigen to the gut lymphoid cells due to their capture and internalization by GALT [10-12]. However,

conventional polymer nanoparticles usually display a low capability to target to a larger extent specific sites within the gastrointestinal tract (i.e. Peyer’s patches), and could be eliminated to some extent by the mucus shed off and intestinal movements [13, 14]. Therefore the immune response elicited with these antigen carriers is usually not so high as necessary to offer the adequate degree of protection to the host [15, 16]. In order to overcome these drawbacks and render nanoparticles more efficient as adjuvants for vaccination, one possible strategy can be their association with compounds or molecules involved in either the colonization process of micro-organisms or the activation of the host immune system. The colonization process of micro-organisms to host tissues involves adherence to the surface of a cell, invasion or internalization in the cell, multiplication and production of extracellular proteins that damage the host cells and facilitate the growth and spread of the pathogen [17-19]. Micro-organisms can invade and colonize the host tissue by using a number of different specific adherence factors including lipoteichoic acids, outer membrane proteins [20], flagellum [21, 22], fimbriae and pili [22, 23], lectins [24] and glycoproteins [25]. Most of these adhesive factors are also considered as immunomodulators and they are included in the generic denomination of PAMPs (pathogen associated molecular patterns). In this context, lipopolysaccharides from gram negative bacteria are well known for their ability to enhance and modulate the host immune response. In previous studies, our group has demonstrated the potential of poly(anhydride) nanoparticles to develop adhesive interactions within the gastrointestinal tract [6, 7, 26] as well as their capability to be easily coated with a number of ligands, including lectins [26], polyethylenglycols [7] or proteins [26, 27]. The aim of this work was to evaluate the real potential as mucosal adjuvants of these nanoparticles associated with two types of micro-organism adhesion factors (flagellin and mannose) in order to mimic the bacterial colonization features. In parallel, we also include the study of the immunomodulator effect of lipopolysaccharide from Brucella ovis when associated with poly(anhydride) nanoparticles. 31

J. DRUG DEL. SCI. TECH., 18 (1) 31-39 2008

Micro-organism-like nanoparticles for oral antigen delivery H.H. Salman, S. Gómez, C. Gamazo, R. Costa Martins, V. Zabaleta, J.M. Irache

Control ovalbumin nanoparticles (OVA-NP1) were prepared in the same way without the use of LPS.

I. Materials and methods 1. Chemicals

Gantrez AN 119 [poly (methyl vinyl ether-co-maleic anhydride)] was kindly donated by International Specialty Products (ISP. Spain). Trypticase soy agar was purchased from Becton Dickinson (Madrid, Spain). Ovalbumin (OVA, Grad V), mannosamine hydrochloride, rhodamine B isothiocyanate (RBITC), 1,3-diaminopropane (1,3-DP) and protease inhibitor cocktail [(4-(2 aminoethyl) benzenesulfonyl fluoride, trans-epoxysuccinyl-leucyl-amido (4 guanidino) butane (E-64), bestatin, leupeptin, aprotinin and sodium EDTA] were purchased from Sigma (Spain). Antibodies peroxidase/conjugate antiIgG1, anti-IgG2a or anti-IgA were supplied by Nordic Immunological Labs (The Netherlands). Salmonella H antiserum poly a-z and heart infusion broth were purchased from Difco (Difco Lab Detroit, USA). All other chemicals used were of analytical grade and obtained from Merck (Spain).

4.2. Preparation of OVA-loaded ligand nanoparticles Flagellin from Salmonella enteritidis and mannosamine were selected to be used as ligands to coat Gantrez AN nanoparticles. Both flagellin (F-NP) and mannosamine (M-NP) coated nanoparticles were prepared according to the protocols previously described [6, 27]. 4.2.1. Preparation of OVA-loaded flagellin nanoparticles Ovalbumin-loaded flagellin nanoparticles (OVA-F-NP) were prepared by a modification of the method described for the preparation of flagellin containing nanoparticles [27]. Briefly, 5 mg of flagellin-enriched extract and 5 mg of ovalbumin were sonicated in 2 mL acetone and then mixed with 3 mL acetone containing 100 mg of Gantrez AN. The organic solution was magnetically stirred for 30 min at room temperature. The polymer was then dissolved by the addition of 10 mL of absolute ethanol followed by 3 mL deionized water containing 5 mg flagellin-enriched extract. The organic solvents were eliminated under reduced pressure (Büchi R-144, Switzerland) and the resulting suspension was magnetically stirred for 1 h at RT. The nanoparticles were cross-linked by incubation with 100 µg 1,3diaminopropane for 5 min, and centrifuged at 27,000 × g for 20 min. Finally, the nanoparticles were lyophilized using sucrose (5%) as cryoprotector. The control nanoparticles OVA-NP2 were prepared as described above without using flagellin-enriched extract.

2. Preparation of rough lipopolysaccharide of Brucella ovis extract (LPS)

To prepare cells for LPS extraction, tryptic soy broth (TSB) flasks were inoculated with fresh cultures of Brucella ovis REO 198 strain, and incubated at 37ºC for 3 days in air under constant shaking. The lipopolysaccharide fraction was obtained from complete cells as described previously using the phenol-chloroform-petroleum ether extraction method [28, 29], and characterized using the thiobarbiturate acid method [30].

4.2.2. Preparation of OVA-loaded mannosylated nanoparticles Briefly, 5 mg of OVA and 1 mg of mannosamine were sonicated and incubated in 5 mL acetone containing 100 mg of Gantrez AN, for 1 h at room temperature. The polymer was then dissolved by the addition of 10 mL of absolute ethanol. For mannosamine coating, 1 mL of deionized water containing 5 mg of mannosamine was added to the organic phase. The organic solvents were eliminated under reduced pressure (Büchi R-144, Switzerland) and the resulting aqueous nanosuspensions were incubated under magnetic stirring for 1 h at RT. OVA-M-NP were purified by centrifugation at 27,000  × g for 20 min and the supernatants were collected to quantify the unbounded mannosamine. Finally, OVA-M-NP nanoparticles were cross-linked by incubation with 100 µg 1,3-diaminopropane for 5 min, and centrifuged at 27,000 × g for 20 min, and lyophilized with sucrose. Control non-mannosylated nanoparticles (OVA-NP2) were prepared without the use of mannosamine.

3. Preparation of the Salmonella enteritidis extract (SE)

The method for isolating and purifying the flagellin-enriched extract (SE) was according to a previous publication [31]. Briefly, the bacterial cells in their stationary phase from cultures on Trypticase soy agar were used to inoculate 800 mL flasks of brain heart infusion broth, and left at 37ºC for 48 h without shaking. The cells were washed with phosphate-buffered saline (PBS; pH 7.4, 10 mmol) and centrifuged (7,000 × g, 30 min). SE bacterial extract was obtained after treatment of the cellular sediment with a chaotropic salt (3M KSCN/PBS) under magnetic stirring (1 h, room temperature), followed by centrifugation (35,000 × g, 30 min). The supernatant containing the flagella-enriched extract was collected, dialyzed against PBS, then deionized water, and finally, lyophilized. Flagellin was characterized and quantified using SDS-PAGE [27].

4. Preparation of Gantrez AN nanoparticles

Gantrez AN nanoparticles were prepared using a solvent displacement method described previously [26]. To study the oral adjuvant capacity of the nanoparticle formulations, ovalbumin (OVA) was used as antigen model to be associated in the nanoparticles.

5. Characterization of nanoparticles

The size and zeta potential of the nanoparticles were determined by photon correlation spectroscopy and electrophoretic laser Doppler anemometry respectively, using a Zetamaster analyzer system (Malvern Instruments, UK). In order to quantify the ovalbumin associated with the nanoparticles, SDS-PAGE was performed by dissolving 5 mg OVA-loaded NP in 2 mL DMF/acetone mixture (1:1 v/v) and then storing it at -20ºC for 24 h. Samples were centrifuged at 10,000 × g for 15 min and the precipitate was washed with cold acetone and centrifuged again. The precipitates were resuspended in sample buffer. Finally, the amount of OVA was estimated by calculating the average band density and in SDS-PAGE using Micro Image software (Version 4.0; Olympus Optical Co., USA) running OVA standard calibration curve in the range between 2.5-0.25 µg/well. The amount of LPS associated to nanoparticles was indirectly estimated by determining one of its exclusive markers, KDO, using the thiobarbiturate acid method [30]. For this purpose, a solution containing digested nanoparticles (NaOH 0.1 N, 24 h, 4ºC) was added to 5 volumes of a solution of methanol and 1% methanol saturated with sodium acetate to precipitate the LPS content. The pellet obtained was

4.1. Preparation of OVA and LPS-entrapped Gantrez AN nanoparticles (OVA-L-NP) Briefly, 5 mg OVA were dispersed in 1 mL acetone by ultrasonication (Microson for 1 min under cooling. Similarly, 1 mg LPS was also dispersed in 1 mL acetone. The OVA and the LPS dispersions were added to 3 mL acetone containing 100 mg Gantrez AN and stirred magnetically for 30 min at room temperature. Then dissolution of the polymer was induced by the addition of 20 mL ethanol: water mixture (1:1 by volume). The organic solvents were eliminated under reduced pressure (Büchi R-144, Switzerland). The resulting nanoparticles were then cross-linked by incubation with 5 µg 1,3-diaminopropane/mg Gantrez AN for 5 min under magnetic stirring at room temperature. Eventually, the cross-linked nanoparticles were fluorescently-labeled by incubation with 1.25 mg of rhodamine B isothiocyanate (RBITC) for 5 min. Finally, the cross-linked LPS nanoparticles were purified by centrifugation and lyophilized using sucrose (5%) as cryoprotector. 32

Micro-organism-like nanoparticles for oral antigen delivery H.H. Salman, S. Gómez, C. Gamazo, R. Costa Martins, V. Zabaleta, J.M. Irache

then resuspended in 0.2% SDS solution and used in the KDO assay. Each sample was assayed in triplicate and results were expressed as the amount of LPS (in µg) per mg nanoparticles. The amount of mannosamine associated to mannosylated nanoparticles was estimated by quantification of mannosamine content in the supernatants collected from the nanoparticle purification step using O-phthalaldehyde (OPA) fluorimetric assay of primary amines [32]. Finally, the amount of flagellin associated with nanoparticles was quantified using SDS-PAGE as described previously [27].

J. DRUG DEL. SCI. TECH., 18 (1) 31-39 2008

9. Immunization protocol

Animal protocols were applied in compliance with the regulations of the responsible committee of the University of Navarre in line with the European legislation on animal experiments (86/609/EU). Female BALB/c mice of average weight (20 ± 1 g) supplied by Harlan (Barcelona, Spain) were divided into 6 groups of 10 mice. Animals were left to fast overnight with free access to water. One single dose of OVA was administered by the oral route (7 groups, n = 10). Animals were immunized by the oral gavage of 200 µL PBS containing equivalent dose of 100 µg OVA in the form of either OVA-F-NP, OVA-M-NP, OVA-NP2 (control nanoparticles) or free OVA. Doses of 25 µg OVA were used in the experiment related to LPS nanoparticles (OVA-L-NP, OVA-NP1 and free OVA).

6. In vivo bioadhesion studies

The bioadhesion studies were carried out using a protocol described previously [26], in compliance with the regulations of the responsible committee of the University of Navarre in line with the European legislation on animal experiments (86/609/EU). Briefly, an aqueous suspension containing 10 mg of the nanoparticles loaded with RBITC (approximately 45 mg particles/ kg body weight) was administered orally to male Wistar rats fasted overnight (average weight 225 g; Harlan, Spain). The animals were sacrificed by cervical dislocation at different times post-administration. The abdominal cavity was opened and the gastrointestinal tract was removed. The gut was then divided into six anatomical regions: stomach (Sto), intestine (I1, I2, I3 and I4) and cecum (Ce). Each mucosa segment was opened lengthwise, rinsed with PBS and digested with 3 N NaOH, for 24 h. RBITC was extracted from the digested samples by the addition of 2 mL methanol, vortexed for 1 min and centrifuged at 2,000 × g for 10 min. Aliquots (1 mL) of the obtained supernatants were diluted with water (3 mL) and assayed for RBITC content by spectrofluorimetry at lex 540 nm and lem 580 nm (GENios, TECAN, Austria) to estimate the fraction of nanoparticles adhering to the mucosa. The standard curves of the bioadhesion study were prepared by addition of RBITC-solutions in 3 N NaOH (0.5-10 µg/mL) to control tissue segments following the same extraction steps (r > 0.996). The experiment was performed in triplicate.

10. Sample collection

Blood samples were collected from the retroorbital plexus during the 6th week post administration. The samples were centrifuged (3,000 × g, 10 min), and the sera were mixed for each group (10 mice). Finally, this pool was diluted 1:10 in PBS and conserved at -80ºC till analysis. Fecal sample collection and analysis was performed as described elsewhere [33]. A pool of fresh pellets from each group of mice was collected into weighed microtubes. Non-fatty milk (3%) in PBS was added at a ratio of 1 mL/100 mg fecal pellets. The pellets were vortexed for 5 min at room temperature. Then the tubes were centrifuged at 10,000 x g for 10 min and the supernatants were transferred to tubes containing 10 µL of protease inhibitor. Finally, pooled samples were saved at -80°C until the time for use.

11. ELISA assay

11.1. Serum anti-OVA IgG1 and IgG2a Specific anti-bodies (IgG1 and IgG2a) against OVA were determined using ELISA with 96 microtiter plates (Thermo LabSystems, Vantaa, Finland). For this purpose, wells were coated with 1 µg OVA in 0.05 M sodium carbonate-bicarbonate buffer (pH 9.6) at 4°C, 24 h and then blocked with 1% BSA in PBS-0.05% Tween 20 (PBS-T) for 1 h at 37°C. After washing with PBS-T, serum samples were added in two-fold serial dilutions in PBS-T starting with 1:40 serum dilution, and incubated at 37°C, for 4 h. Washed wells were then incubated for 2 h, 37°C with antibodies peroxidase/conjugate anti-IgG1 or anti-IgG2a The substrate-chromogen used was H2O2-ABTS (3-ethylbenzthiazoline-6-sulfonic acid). The absorbance (Abs.) was determined at lmax 405 nm (iEMS Reader MF by Labsystems, Vantaa, Finland). The end titers were determined as the dilution of sample giving the mean Abs. ≥ 0.2 the obtained from untreated mice sera.

7. Parameters of bioadhesion

For each nanoparticle formulation, the total adhered fraction of the nanoparticles in the entire gastrointestinal tract was plotted versus time. From these curves the following bioadhesion parameters: Qmax, AUCadh, MRTadh and Kadh were estimated from 0 to 8 h post-administration as described previously [26] and calculated using WinNonlin 1.5 software (Pharsight Corporation, USA). Qmax (mg) is the maximum amount of nanoparticles adhering to the entire gut surface (for a given time, Tmax, the sum of the adhered fraction in all the gut portions) and is related to the capacity of the material to develop adhesive interactions. The AUCadh (mg.h) is the area under the curve of the adhered nanoparticles which was calculated using the trapezoidal rule up to tz, and represented the intensity of the bioadhesion. MRTadh (h) is the mean residence time of the adhered fraction of the nanoparticles to the mucosa. Kadh was defined as the terminal elimination rate of the adhered fraction in the mucosa.

11.2. Fecal anti-OVA IgA After the washing step of OVA-coated plates as described above, the plates were blocked with 200 µL 3% non-fatty milk in PBS-T for 1 h, at room temperature. Fecal extract samples of 100 µL were added starting with an undiluted one and followed by twofold serial dilutions till 1:64 in PBS-T, and the plates were incubated at 37ºC, for 4 h. Finally, washed wells were treated with anti-body peroxidase/ conjugates anti-IgA (GAM/IgA/PO) 1:1000 dilution. The detection step and the end titers were determined as described above.

8. Tissue fluorescence microscopy studies

The distribution of RBITC-loaded mannosylated nanoparticles in the gastrointestinal normal mucosal tissue and Peyer’s patches was visualized by fluorescence microscopy. For this purpose 10 mg of RBITC-labeled nanoparticles were orally administered to rats as described above. The animals were sacrificed 2 h later and the ileum was removed and washed with PBS. Mucosal portions from the ileum of about 0.5 cm length were then treated with the tissue-embedding medium OCT (Sakura, The Netherlands) and frozen in liquid nitrogen. Tissue samples were cut into 5 µm longitudinal sections in a cryostat (2800 Frigocut E, Reichert-Jung, Germany), attached to poly-L-lysine precoated slides (Sigma, Madrid, Spain) and stored at -20ºC before fluorescence microscopic visualization.

12. Statistical analysis

The bioadhesion data and the physicochemical characteristics were compared using the nonparametric Mann-Whitney U-test and Student t-test respectively. P values of < 0.05 were considered significant. All calculations were performed using SPSS statistical software program (SPSS 10, Microsoft, USA).

33

J. DRUG DEL. SCI. TECH., 18 (1) 31-39 2008

Micro-organism-like nanoparticles for oral antigen delivery H.H. Salman, S. Gómez, C. Gamazo, R. Costa Martins, V. Zabaleta, J.M. Irache

II. Results 1. Evaluation of the encapsulation of LPS on Gantrez AN nanoparticles

A 3

1.1. Physicochemical characterization The main physicochemical characteristics of Gantrez AN formulations are summarized in Table I. For LPS-loaded nanoparticles, the amount of LPS incorporated in the nanoparticles was about 14 µg/ mg NP. On the other hand, the presence of LPS did not influence the mean size (about 230 nm) or protein loading (about 30µg/mg) of OVA-loaded nanoparticles (OVA-NP1 and OVA-L-NP). In contrast, the presence of LPS significantly decreased the negative zeta potential of OVA-loaded nanoparticles (-34 mV vs -50 mV).

Adhered amount (mg)

2.5 2 1.5 1 0.5

Sto

Table I - Influence of the LPS on the physicochemical characteristics of Gantrez AN nanoparticles. Data represent the mean ± SD (n = 10). OVA content (µg/mg)

158 ± 3 239 ± 4* 227 ± 4*

-45.1 ± 0.5 -50.8 ± 2.9 -34.1 ± 3.4

13.8 ± 3.0

30.1 ± 4.5 26.5 ± 0.3

I3

I4

NP Ce

3 2.5

NP: empty nanoparticles. OVA-NP1: OVA-entrapped nanoparticles. OVA-L-NP: OVA and LPS-entrapped nanoparticles. *p < 0.05 using Student-t test.

1.2. In vivo bioadhesion studies Figure 1 shows the distribution of the adhered amounts of nanoparticles in the gut mucosa 1 and 3 h post-administration. Overall OVA-nanoparticle formulations appeared to interact with the mucosa to a greater extent than control nanoparticles (NP). OVA-NP1 displayed a maximum of adhesion in the stomach, where about 19% of the given dose was found adhered 1 h post-administration. Interestingly, about 20% of the loaded dose of these nanoparticles was also found adhered to the small intestine (mainly duodenum and jejunum, portions I1-I3 in Figure 1) during at least the first 3 h post-administration. On the other hand, the presence of LPS modified the distribution of OVA nanoparticles within the gut. Thus OVA-L-NP displayed two maximums of adhesion: in the stomach (about 12% of the given dose 1 h after administration) and in the duodenum (11% of the given dose 3 h post-administration). In this context, 3 h post-administration, OVA-LNP displayed adhesion that was twice as high in I1 than OVA-NP1. Figure 2 shows the parameters of bioadhesion (Qmax, AUCadh, MRTadh and Kadh) calculated from the curves of bioadhesion (see Materials and methods). The capability of the nanoparticles to develop adhesive interactions within the gut (expressed as Qmax), and the intensity of the phenomena (expressed as AUCadh) were found to be twice as high for OVA loaded nanoparticles than for control ones (NP) (p < 0.01). However, OVA-L-NP displayed a similar adhesive potential than the same formulation without LPS. On the other hand, the elimination rate of the adhered fraction (Kadh) of the different formulations tested was found to be quite similar and close to 0.10 h-1. Similarly the mean residence time of the adhered fraction of nanoparticles was calculated to be close to 3.5 h. Figure 3 shows fluorescence microscopy images of ileum samples from the animals treated with nanoparticle formulations fluorescently labeled with RBITC. The nanoparticles were visualized in the gut as red fluorescent spots. Control nanoparticles (NP) displayed a low ability to adhere to the normal mucosal tissue (Mucosa site Figure 3A) including Peyer’s patches (PP). In contrast, OVA-L-NP were found adhered to the normal mucosal tissue of the ileum, and demonstrated a slight adhesion to the mucosal surface of Peyer’s patches. Interestingly, OVA-NP1 showed a strong capability to adhere and penetrate Peyer´s patches (PP), whereas for OVA-L-NP this phenomenon was not observed.

2 1.5 1 0.5 OVA-L-NP OVA-NP1

0 Sto

I1

I2

I3

I4

NP Ce

Figure 1 - Gut distribution of nanoparticle formulations in the different segments of the gastrointestinal tract at 1 (A) and 3 h (B) postadministration. Sto: Stomach; I1, I2, I3 and I4: consecutive portions of the small intestine; Ce: Cecum. The formulations were OVA and LPS entrapped nanoparticles (OVA-L-NP1); OVA-entrapped nanoparticles (OVA-NP1); and control nanoparticles (NP). a)

**

Qmax (mg)

7

*

b)

*

35

6

30

5

25

AUCadh (mg h)

LPS content (µg/mg)

I2

4 3 2 1

*

20 15 10 5

0

0

OVA-NP1

OVA-L-NP

NP

c) 4.0

0.20

OVA-NP1

OVA-L-NP

NP

OVA-NP1

OVA-L-NP

NP

d)

3.5 3.0

0.15 -1

Kadh (h )

2.5

MRT (h)

Zeta potential (mV)

OVA-NP1

I1

B

Adhered amount (mg)

NP OVA-NP1 OVA-L-NP

Size (nm)

OVA-L-NP

0

0.10

2.0 1.5 1.0

0.05

0.5 0.0

0.00

OVA-NP1

OVA-L-NP

NP

Figure 2 - Parameters of bioadhesion: a) Qmax: maximal amount of nanoparticles adhered to the gut surface; b) AUCadh: area under the curve of bioadhesion; c) Kadh: terminal elimination rate of the adhered fraction in the gastrointestinal mucosa; and d) MRT: mean residence time of the adhered fraction of nanoparticles in the mucosa. Formulations: OVA entrapped nanoparticles (OVA-NP1); OVA and LPS entrapped nanoparticles (OVA-L-NP) and control nanoparticles (NP).

1.3. Immune response Figure 4 shows the antibody titers (IgG2a and IgG1) on day 42 after oral immunization with a single dose equivalent to 25µg OVA in OVA-L-NP, OVA-NP1 and control ovalbumin solution (OVA). In this 34

Micro-organism-like nanoparticles for oral antigen delivery H.H. Salman, S. Gómez, C. Gamazo, R. Costa Martins, V. Zabaleta, J.M. Irache

J. DRUG DEL. SCI. TECH., 18 (1) 31-39 2008

A 3

Adhered amount (mg)

2.5

Figure 3 - Visualization of control nanoparticles (NP) (A); OVA entrapped nanoparticles (OVA-NP1) (B); and OVA and LPS entrapped nanoparticles (OVA-L-NP) (C) in the normal mucosa (M) of the ileum and the follicle-associated epithelium of Peyer’s patches (PP) by fluorescence microscopy.

2 1.5 1 0.5 F-NP M-NP

0

OVA dose: 25 µg

Sto

Th1 (IgG 2a ) Th2 (IgG1 )

I1

I2

I3

I4

B

Ce

NP

OVA-L-NP 3 2,5 Adhered amount (mg)

OVA-NP1

OVA

0

1

2

3

4

5

6

7

8

9

Log 2 titer

2 1,5 1 0,5 F-NP

0 Sto

Figure 4 - Serum OVA-specific IgG2a and IgG1 in BALB/c mice (n =10) 6 weeks post immunization with 25 µg OVA in OVA-L-NP and OVA-NP1 and OVA solution as a control.

I1

M-NP I2

I3

I4

NP Ce

Figure 5 - Gut distribution of nanoparticle formulations in the different segments of the gastrointestinal tract at 1 (A) and 3 h (B) postadministration. Sto: Stomach; I1, I2, I3 and I4: consecutive portions of the small intestine; Ce: Cecum. The formulations were flagellin nanoparticles (F-NP); mannosylated nanoparticles (M-NP); and control nanoparticles (NP).

case, both nanoparticle formulations induced higher levels of both IgG1 and IgG2a than the control (OVA). However, the enhancement of the immune response was considerably higher for the group treated with OVA-entrapped nanoparticles without LPS than for OVA-L-NP.

2.2. In vivo bioadhesion studies Figure 5 shows the distribution of the adhered amounts of nanoparticles in the gut mucosa 1 and 3 h post-administration. Starting a gross comparison among bioadhesion profiles of all nanoparticle formulations, it can be deduced that there is a significant increase of the bioadhesive capacity of surface modified nanoparticles with flagellin or mannosamine compared to control ones at 1 and 3 h post-administration. In this context both types of nanoparticles demonstrated a high tropism in the lower parts of the gut (mainly the terminal jejunum and ileum, fractions: I3 and I4), which represented about 45 and 30% of the total adhered fraction within the entire gastrointestinal tract gut for both F-NP and M-NP. From the calculation of the parameters of bioadhesion (Figure 6), it is clear that F-NP and M-NP displayed a higher potential to develop adhesive interactions within the gut than control nanoparticles.

2. Evaluation of the coating of Gantrez AN nanoparticles with ligands

2.1. Characterization of OVA-loaded ligand nanoparticle conjugates Table II summarizes the main physicochemical of OVA-loaded nanoparticles. Both OVA-ligand nanoparticles (OVA-F-NP and OVAM-NP) displayed a homogeneous size ranging from 350-400 nm, which was significantly higher than OVA-loaded conventional nanoparticles (OVA-NP2). The presence of flagellin or mannosamine on the surface of the nanoparticles significantly decrease the surface negative charge compared to control nanoparticles. For OVA-F-NP the amount of flagellin content was calculated to be about 12 µg/mg whereas for OVA-M-NP, the amount of ligand was about 30 µg mannosamine per mg nanoparticles. Finally, the presence of flagellin or mannosamine slightly decreased the amount of OVA in the nanoparticles.

Table II - Influence of the ligands on the physicochemical characteristics of Gantrez AN nanoparticles. Data represent the mean ± SD (n = 10). NP OVA-NP2 OVA-F-NP OVA-M-NP

Size (nm)

Zeta potential (mV)

OVA content (µg/mg)

Mannosamine content (µg/mg)

Flagellin content (µg/mg)

158 ± 3 277 ± 13 391 ± 5* 350 ± 1*

-45.1 ± 0.6 -48.2 ± 4.3 -34.3 ± 2.2* -44.2 ± 1.3*

11.9 ± 1.5 7.3 ± 2.4** 9.1 ± 2.3

32 ± 1

11.6 ± 0.6 -

OVA-NP2: ovalbumin-loaded control nanoparticles. OVA-F-NP: ovalbumin-loaded flagellin coating nanoparticles. OVA-M-NP: ovalbumin-loaded mannosamine-coated nanoparticles. *p < 0.05 to compare size and zeta potential of OVA-ligand nanoparticles (OVA-F-NP, OVA-M-NP) with OVA-NP2 using Student-t test. **p < 0.05 using Student-t test to compare ovalbumin content of OVA-F-NP with both OVA-NP2 and OVA-M-NP using Student-t test. 35

J. DRUG DEL. SCI. TECH., 18 (1) 31-39 2008

**

a) 6

**

b)

*

OVA dose: 100 µg

*

30

5

OVA-M-NP

25

AUCadh (mg h)

4

Qmax (mg)

Micro-organism-like nanoparticles for oral antigen delivery H.H. Salman, S. Gómez, C. Gamazo, R. Costa Martins, V. Zabaleta, J.M. Irache

3

2

1

20 15

OVA-F-NP

10 5 0

0

F-NP

c)

M-NP

F-NP

NP

M-NP

OVA-NP2

NP

d)

*

4.0

0.25

OVA

3.5 3.0

0.20

MRT (h)

-1

Kadh (h )

2.5 0.15

0.10

0

2.0

1

2

3

4

5

6

7

Log 2 titer

1.5 1.0

0.05

Figure 9 - Fecal OVA-specific IgA 6 weeks post oral immunization by a single dose of the ligand coated nanoparticles formulations containing 100 µg OVA for OVA-M-NP, OVA-F-NP, OVA-NP2 and free OVA.

0.5 0.00

0.0

F-NP

M-NP

NP

F-NP

M-NP

NP

Figure 6 - Parameters of bioadhesion: a) Qmax: maximal amount of nanoparticles adhered to the gut surface; b) AUCadh: area under the curve of bioadhesion; c) Kadh: terminal elimination rate of the adhered fraction in the gastrointestinal mucosa; and d) MRT: mean residence time of the adhered fraction of nanoparticles in the mucosa. Formulations: flagellin nanoparticles (F-NP); mannosylated nanoparticles (M-NP) and control nanoparticles (NP).

fraction of ligand-nanoparticles was 30 min shorter than for control nanoparticles. Figure 7 shows fluorescence microscopy images of ileum samples from the animals treated with 10 mg of nanoparticles fluorescently RBITC-labeled mannosamine, flagellin nanoparticles (M-NP, FNP) and control ones (NP). Control nanoparticles (NP) showed a low affinity for both normal mucosal layer and Peyer`s patches (Figure 7A). In contrast, both mannosylated and flagellin nanoparticles demonstrated a strong capacity to adhere to or interact with the normal mucosal tissue and to be internalized by Peyer’s patches (Figure 7B and 7C). 2.3. Immune response Figure 8 shows the antibody titers (IgG2a and IgG1) on day 42 after oral immunization with a single dose of 100 µg OVA loaded in flagellin nanoparticles (OVA-F-NP), mannosamine nanoparticles (OVA-M-NP) and control ones (OVA-NP2). Both OVA-loaded ligand nanoparticle conjugates (OVA-M-NP, or OVA-F-NP), induced a significant enhancement of the immune responses after oral immunization compared to control nanoparticles (OVA-NP2). In this context, oral immunization with control nanoparticles showed a low and Th2 predominant immune response. However, for animals immunized with OVA-F-NP, the blood level of IgG2a (Th1) and IgG1 (Th2) was similar. The oral delivery of OVA loaded in ligand-nanoparticles significantly enhanced the mucosal immune response compared with control nanoparticles or free OVA. This mucosal immune response (IgA) was six and seven times higher than the control in the case of OVA-F-NP and OVA-M-NP respectively (Figure 9).

Figure 7 - Visualization of control nanoparticles (NP) (A); mannosylated nanoparticles (M-NP) (B); and flagellin nanoparticles (F-NP) (C) in the normal mucosa (M) of the ileum and the follicle-associated epithelium of Peyer’s patches (PP) by fluorescence microscopy.

Th1 (IgG2a ) Th2 (IgG1)

OVA dose: 100 µg OVA-M-NP

OVA-F-NP

OVA-NP2

III. Discussion

Nanoparticles have been proposed as mucosal adjuvants for oral vaccination; however, in some cases, their ability to increase or modulate the immune response by the host is poor. In principle, these polymer carriers offered a number of advantages including protection of the loaded antigen against its gut degradation or inactivation and controlled release properties. Nevertheless, the ability of polymer nanoparticles to develop adhesive interactions within the gut and, much more important for vaccination, their capability to target and specifically interact with components of GALT is sometimes mediocre. In this context, one possible way of improving the adjuvant effect of nanoparticles may be their association with compounds or molecules able to try to copy or imitate closely the bacteria and virus behavior, concerning the strategies developed by these micro-organisms during their evolution to invade and interact with the immune cells of a host.

OVA 0

1

2

3

4

5

6

7

Log 2 titer

Figure 8 - Serum OVA-specific IgG2a and IgG1 in BALB/c mice (n =10) 6 weeks post immunization with 100 µg OVA in OVA-M-NP, OVA-F-NP and OVA-NP2.

Thus Qmax was found to be 2.5 times higher for F-NP and M-NP than NP. Similarly, the intensity of the adhesive phenomenon (AUC) described for M-NP and F-NP was at least 2 and 3 times higher than that calculated for NP. In contrast, the adhered fraction of F-NP was eliminated from the mucosa faster than M-NP or NP (p < 0.05). Finally, it is interesting to note that the mean residence time of the adhered 36

Micro-organism-like nanoparticles for oral antigen delivery H.H. Salman, S. Gómez, C. Gamazo, R. Costa Martins, V. Zabaleta, J.M. Irache

So the idea of developing micro-organism-like nanoparticles or biomimetic nanoparticles may involve at least two different strategies. The former would be to mimic the ability of micro-organisms to colonize and invade a given cell. In fact, the phenomenon of micro-organism adhesion to the surface of a cell is the first step and a prerequisite for the colonization and invasion of the host. During their evolution, bacteria have developed a number of strategies including the use of adhesins (i.e. flagella) and glycoconjugates (i.e. mannose proteins). The second strategy would be the association of PAMPs, such as lipopolysaccharide, as immunopotentiator or immunomodulator. In the first case, the association of adhesins and glycoconjugates to the antigen-loaded nanoparticles would improve the immune response, making it possible to simplify the vaccination regime. In the second case, the presence of the immunomodulator (i.e. LPS) would make it possible to shift the immune response to obtain a better effect from the immune cells (i.e. to potentiate a cellular or a humoral response). In order to mimic gut colonization and/or the immunoadjuvant effect of micro-organisms, we proposed two possible alternatives in this work. The first was the incorporation of lipopolysaccharide from Brucella ovis into Gantrez AN nanoparticles. The second was to coat the surface of the nanoparticles with adhesive ligands, either mannosamine or flagellin from Salmonella enteritidis. In all cases, the adjuvant capability for vaccination of these micro-organism-like nanoparticles was evaluated using ovalbumin as an antigen model.

J. DRUG DEL. SCI. TECH., 18 (1) 31-39 2008

phenomenon would be the reason why the LPS improved neither the gut adhesion of nanoparticles (UF, UF) nor the immune response and thus the association of LPS with the nanoparticles did not affect the efficacy of the formulations.

2. Evaluation of coating of Gantrez AN nanoparticles with ligands

Flagellin from Salmonella enteritidis is considered as the key element forming the typical flagella of this micro-organism. This protein is about 53 kDa and is encoded by the fliC gene [44]. Studies have described the importance of the flagella in salmonella invasion and colonization [21, 45]. On the other hand, mannosamine has been used due to the data available about the implication of mannose residues expressed on the surface of some micro-organisms such as Candida albicans in their adhesion and colonization of the mucosal cells [46]. This adhesive mechanism is mediated by the high affinity binding of mannose to the so-called mannose-binding proteins which specifically recognizes carbohydrate moieties, terminated with mannose, on the surface of pathogens [47]. For both types of ligand-nanoparticles, a significant increase of the bioadhesive capability was clearly noted compared with the control (Figure 5). In fact, flagellin-coated and mannosamine-coated nanoparticles demonstrated a high tropism for the lower parts of the gut (I3 and I4), which represented about 45 and 30% of the total adhered fraction within the whole segments of the gut for both F-NP and M-NP respectively. In the case of flagellin nanoparticles, the ileum tropism and colonization profile of these nanoparticles was approximately similar to that described for Salmonella cells in the same animal model [45]. In contrast, the strong adhesive interactions of mannosylated nanoparticles may be related to the high binding affinity of mannose residues for the mannose-binding lectins (MBL) which are expressed on the lymphoid and non-lymphoid cells of the gut [47]. The kinetic parameters of bioadhesion (Figure 6), M-NP and F-NP displayed a higher ability to develop adhesive interactions (Qmax) than control nanoparticles (NP). Similarly, AUCadh, clearly demonstrated the higher bioadhesion intensity of those conjugates compared to non-coated nanoparticles. In contrast, the elimination rate of the adhered fraction (Kadh) for F-NP was found to be higher than for the adhered fraction of M-NP or control nanoparticles, indicating a faster elimination from the mucosa. On the other hand, fluorescence microscopy indicated the stronger affinity of both ligand nanoparticle conjugates to both normal mucosal and lymphoid tissue (Peyer’s patches) than control nanoparticles (Figure 7). Both ligands facilitated the penetration of the nanoparticles to both normal and lymphoid tissues which gave them the possibility to be used as non-live micro-organisms like polymeric vectors in oral delivery systems. In any case, from these photographs it is clear that both types of ligand nanoparticle are able to interact, penetrate and cross the mucosa. Oral immunization with ligand nanoparticle conjugate (OVA-F-NP and OVA-M-NP) formulations induced stronger and more balanced serum titers of IgG2a (Th1) and IgG1 (Th2) than control nanoparticles which induced a typical Th2 response (Figure 8). This Th1 response enhancement may be related to the high tropism of flagellin or mannosamine nanoparticles to the distal regions of the intestine (ileum) and uptake by Peyer’s patches [6, 48]. This may be correlated with a previous study indicating that the delivery of OVA-loaded microbeads to lower intestine region enhances Th1 response [49]. In fact, Th1 enhancement induced by OVA-loaded flagellin nanoparticles (OVA-F-NP) response may be related to the effective uptake and activation of antigen presenting cells (DCs) by flagellin via TLR-5 (Toll like receptor-5). Most TLR agonists function as adjuvants by stimulating the production of cytokines and the maturation of dendritic cells, thereby linking innate and adaptive immunity. Flagellin from gram-negative organisms signals via TLR5 and has effects on both

1. Evaluation of the encapsulation of LPS on Gantrez AN nanoparticles

The use of LPS as immunomodulator has been proposed previously [34, 35]. In fact, LPS is known to potentiate Th1 responses and, for this property, it has been incorporated into some vehicles such as microparticles [36] or nanoparticles [37, 38] in order to enhance their adjuvant effect. However, a main drawback when using LPS is its intrinsic toxicity [39, 40]. Therefore, in this investigation a rough lipopolysaccharide from Brucella ovis was selected. This LPS is known to show a low endotoxic effect [41] and to enhance Th1-like responses [5]. For LPS nanoparticles, we could observe that the presence of LPS did not influence either the size or the OVA loading of the resulting carriers. Similarly, the presence of LPS did not negatively influence either the distribution of nanoparticles within the gut or the ability of Gantrez AN to develop adhesive interactions within the gut (Figures 1 and 2). In fact, OVA-NP1 and OVA-L-NP displayed a similar bioadhesive profile and the adhered fraction of nanoparticles from these two formulations was eliminated at the same rate, displaying a similar Kadh and MRTadh. Surprisingly, the OVA-NP1 formulation showed a stronger capability to adhere and penetrate in Peyer’s patch regions than LPS-nanoparticles as visualized by fluorescence microscopy (Figure 3). On the other hand, when these formulations were orally administered to mice, the only formulation that really enhanced the immune response was OVA-NP1. Surprisingly, the presence of the LPS on the NP (OVA-L-NP) decreased both OVA-specific IgG2a (Th1 marker) and IgG1 (Th2 marker) levels (Figure 4). This fact did not correlate with the data previously obtained using the intradermal route (unpublished data). These results appear to contradict those described by other authors who have incorporated lipopolysaccharide or lipid A (which is known to be the region of the lipopolysaccharide responsible for its adjuvant capacity [42]) into some vehicles such as microparticles [36] or nanoparticles [37, 38] in order to enhance their adjuvant effect. All these surprising findings and contradictions could be related to Otte’s hypothesis which recently proposed that the continuous presence of LPS from the different bacteria in the gastrointestinal tract induces a status of hyporesponsiveness and down-regulation of the Toll-like receptor (TLR) cell surface expression [43]. This 37

J. DRUG DEL. SCI. TECH., 18 (1) 31-39 2008

Micro-organism-like nanoparticles for oral antigen delivery H.H. Salman, S. Gómez, C. Gamazo, R. Costa Martins, V. Zabaleta, J.M. Irache

innate and adaptive immune responses [50, 51], being described as activating DCs releasing cytokines to produce a Th1 response [52]. In the case of oral immunization with OVA-loaded mannosylated nanoparticles (OVA-M-NP), the higher IgG2a levels (Th1 response) are related to the mannosylation strategy of the nanoparticles. In correlation, the successful enhancement of Th1 cytokine (IL-12, INF-g) secretion after parenteral administration of mannosylated cationic liposomes (Man liposome/pCMV-OVA) [53] or mannan-coated liposome-protamine-DNA (LPD) nanoparticles has been reported [54]. In addition it has been found that mannose receptors mediate more efficient endocytosis of mannosylated peptides to dendritic cells (DCs) than non-mannosylated ones [55]. However, peptide mannosylation strategy or particulate systems still demonstrate little success in oral antigen delivery compared to parenteral ones. Finally, oral immunization with OVA-F-NP or OVA-M-NP induced a higher mucosal IgA response which was at least 6-7 titers more than control ones (Figure 9). In fact, this strong mucosal immune response was not noted in the case of SC administration (data not shown). Again, this phenomenon is another evidence of the effective uptake of flagellin or mannosamine nanoparticles by gut Peyer’s patches and the passage of the particulate system to lymphocytes causing an effective generation of mucosal IgA. This fact is in agreement with previous observations describing the oral adjuvant effect of mannosylated niosomes that induce a stronger IgA response compared to parenteral forms [56].

8.

9.

10.

11.

12.

13. 14.

*

15.

In summary, the present results demonstrate the viability of trying to imitate the strategies of micro-organisms to adhere to the surface of the gut mucosa and thus to improve the mucosal (oral) adjuvant effect of nanoparticles. The bioadhesive capacity and ileal tropism of the orally administered flagellin- or mannosamine-coated nanoparticles appeared to be instrumental for the effective elicitation of both systemic and mucosal immune responses. In contrast, under the experimental conditions described here, the association of the LPS of Brucella ovis to nanoparticles did not improve the mucosal adjuvant effect of conventional nanoparticles. Further efforts should be focused on exploring this negative effect observed by the oral administration of LPS-nanoparticles.

16. 17. 18. 19. 20.

References 1.

2. 3.

4.

5.

6. 7.

21.

Walker S.M., Pajno G.B., Lima M.T., Wilson D.R., Durham S.R. - Grass pollen immunotherapy for seasonal rhinitis and asthma: a randomized, controlled trial. - J Allergy Clin Immunol, 107, 87-93, 2001. O'Hagan D.T., Singh M. - Microparticles as vaccine adjuvants and delivery systems. - Expert Rev Vaccines, 2, 269-283, 2003. Gomez S., Gamazo C., Roman B.S., Ferrer M., Sanz M.L., Irache J.M. - Gantrez AN nanoparticles as an adjuvant for oral immunotherapy with allergens. - Vaccine, 25, 5263-5271, 2007. Jain S., Sharma R.K., Vyas S.P. - Chitosan nanoparticles encapsulated vesicular systems for oral immunization: preparation, in vitro and in vivo characterization. - J Pharm Pharmacol, 58, 303-310, 2006. Murillo M., Goni M.M., Irache J.M., Arangoa M.A., Blasco J.M., Gamazo C. - Modulation of the cellular immune response after oral or subcutaneous immunization with microparticles containing Brucella ovis antigens. - J Control Release, 85, 237-246, 2002. Salman H.H., Gamazo C., Campanero M.A., Irache J.M. - Bioadhesive mannosylated nanoparticles for oral drug delivery. - J Nanosci Nanotechnol, 6, 3203-3209, 2006. Yoncheva K., Guembe L., Campanero M.A., Irache J.M. -

22. 23.

24. 25. 26. 27. 28.

38

Evaluation of bioadhesive potential and intestinal transport of pegylated poly(anhydride) nanoparticles. - Int J Pharm, 334, 156-165, 2007. Galindo-Rodriguez S.A., Allemann E., Fessi H., Doelker E. Polymeric nanoparticles for oral delivery of drugs and vaccines: a critical evaluation of in vivo studies. - Crit Rev Ther Drug Carrier Syst, 22, 419-464, 2005. Ochoa J., Irache J.M., Tamayo I., Walz A., DelVecchio V.G., Gamazo C. - Protective immunity of biodegradable nanoparticlebased vaccine against an experimental challenge with Salmonella enteritidis in mice. - Vaccine, 25, 4410-4419, 2007. McClean S., Prosser E., Meehan E., O'Malley D., Clarke N., Ramtoola Z., Brayden D. - Binding and uptake of biodegradable poly-DL-lactide micro- and nanoparticles in intestinal epithelia. - Eur. J. Pharm. Sci, 6, 153-163, 1998. Van Der Lubben I.M., Konings F.A., Borchard G., Verhoef J.C., Junginger H.E. - In vivo uptake of chitosan microparticles by murine Peyer's patches: visualization studies using confocal laser scanning microscopy and immunohistochemistry. - J. Drug. Target, 9, 39-47, 2001. Coppi G., Iannuccelli V., Sala N., Bondi M. - Alginate microparticles for Polymyxin B Peyer's patches uptake: microparticles for antibiotic oral administration. - J. Microencapsul, 21, 829-839, 2004. Ponchel G., Irache J. - Specific and non-specific bioadhesive particulate systems for oral delivery to the gastrointestinal tract. - Adv. Drug. Deliv. Rev, 34, 191-219, 1998. Lavelle E.C. - Targeted delivery of drugs to the gastrointestinal tract. - Crit. Rev. Ther. Drug. Carrier. Syst, 18, 341-386, 2001. Carcaboso A.M., Hernandez R.M., Igartua M., Rosas J.E., Patarroyo M.E., Pedraz J.L. - Potent, long lasting systemic antibody levels and mixed Th1/Th2 immune response after nasal immunization with malaria antigen loaded PLGA microparticles. - Vaccine, 22, 1423-1432, 2004. Hori M., Onishi H., Machida Y. - Evaluation of Eudragit-coated chitosan microparticles as an oral immune delivery system. - Int J Pharm, 297, 223-234, 2005. Conway B., Ronald A. - An overview of some mechanisms of bacterial pathogenesis. - Can J Microbiol, 34, 281-286, 1988. Freter R. - Mechanisms of association of bacteria with mucosal surfaces. - Ciba Found Symp, 80, 36-55, 1981. Sansonetti P.J. - Bacterial pathogens, from adherence to invasion: comparative strategies. - Med Microbiol Immunol, 182, 223-232, 1993. Fadl A.A., Venkitanarayanan K.S., Khan M.I. - Identification of Salmonella enteritidis outer membrane proteins expressed during attachment to human intestinal epithelial cells. - J Appl Microbiol, 92, 180-186, 2002. Allen-Vercoe E., Woodward M.J. - The role of flagella, but not fimbriae, in the adherence of Salmonella enterica serotype Enteritidis to chick gut explant. - J Med Microbiol, 48, 771-780, 1999. Darwin K.H., Miller V.L. - Molecular basis of the interaction of Salmonella with the intestinal mucosa. - Clin Microbiol Rev, 12, 405-428, 1999. Humphries A.D., Townsend S.M., Kingsley R.A., Nicholson T.L., Tsolis R.M., Baumler A.J. - Role of fimbriae as antigens and intestinal colonization factors of Salmonella serovars. - FEMS Microbiol Lett, 201, 121-125, 2001. Kaltner H., Stierstorfer B. - Animal lectins as cell adhesion molecules. - Acta Anat (Basel), 161, 162-179, 1998. Lloyd D.H., Viac J., Werling D., Reme C.A., Gatto H. - Role of sugars in surface microbe-host interactions and immune reaction modulation. - Vet Dermatol, 18, 197-204, 2007. Arbos P., Arangoa M.A., Campanero M.A., Irache J.M. - Quantification of the bioadhesive properties of protein-coated PVM/ MA nanoparticles. - Int J Pharm, 242, 129-136, 2002. Salman H.H., Gamazo C., Campanero M.A., Irache J.M. Salmonella-like bioadhesive nanoparticles. - J. Control. Release, 106, 1-13, 2005. Gamazo C., Winter A.J., Moriyon I., Riezu-Boj J.I., Blasco J.M., Diaz R. - Comparative analyses of proteins extracted by hot saline or released spontaneously into outer membrane blebs

Micro-organism-like nanoparticles for oral antigen delivery H.H. Salman, S. Gómez, C. Gamazo, R. Costa Martins, V. Zabaleta, J.M. Irache

29. 30. 31.

32.

33.

34. 35. 36.

37.

38.

39.

40. 41.

42. 43.

44.

from field strains of Brucella ovis and Brucella melitensis. - Infect Immun, 57, 1419-1426, 1989. Galanos C., Luderitz O., Westphal O. - A new method for the extraction of R lipopolysaccharides. - Eur J Biochem, 9, 245249, 1969. Warren L. - The thiobarbituric acid assay of sialic acids. - J Biol Chem, 234, 1971-1975, 1959. Ochoa-Reparaz J., Sesma B., Alvarez M., Jesus Renedo M., Irache J.M., Gamazo C. - Humoral immune response in hens naturally infected with Salmonella enteritidis against outer membrane proteins and other surface structural antigens. - Vet Res, 35, 291-298, 2004. Benson J.R., Hare P.E. - O-phthalaldehyde: fluorogenic detection of primary amines in the picomole range. Comparison with fluorescamine and ninhydrin. - Proc Natl Acad Sci USA, 72, 619-622, 1975. Maciel M., Fusaro A.E., Oliveira C.R., Futata E.A., Duarte A.J., Sato M.N. - IgA response in serum and gut secretion in sensitized mice fed with the dust mite Dermatophagoides pteronyssinus extract. - Braz J Med Biol Res, 37, 817-826, 2004. Cox E., Verdonck F., Vanrompay D., Goddeeris B. - Adjuvants modulating mucosal immune responses or directing systemic responses towards the mucosa. - Vet Res, 37, 511-539, 2006. Dzierzbicka K., Kolodziejczyk A.M. - [Adjuvants - essential components of new generation vaccines]. - Postepy Biochem, 52, 204-211, 2006. Kazzaz J., Singh M., Ugozzoli M., Chesko J., Soenawan E., O'Hagan D T. - Encapsulation of the immune potentiators MPL and RC529 in PLG microparticles enhances their potency. - J Control Release, 110, 566-573, 2006. Diwan M., Elamanchili P., Lane H., Gainer A., Samuel J. - Biodegradable nanoparticle mediated antigen delivery to human cord blood derived dendritic cells for induction of primary T cell responses. - J Drug Target, 11, 495-507, 2003. Cui Z., Mumper R.J. - The effect of co-administration of adjuvants with a nanoparticle-based genetic vaccine delivery system on the resulting immune responses. - Eur J Pharm Biopharm, 55, 11-18, 2003. Goldstein J., Hoffman T., Frasch C., Lizzio E.F., Beining P.R., Hochstein D., Lee Y.L., Angus R.D., Golding B. - Lipopolysaccharide (LPS) from Brucella abortus is less toxic than that from Escherichia coli, suggesting the possible use of B. abortus or LPS from B. abortus as a carrier in vaccines. - Infect Immun, 60, 1385-1389, 1992. Jones L.M., Diaz R., Berman D.T. - Endotoxic activity of rough organisms of Brucella species. - Infect Immun, 13, 1638-1641, 1976. Weiss D.S., Takeda K., Akira S., Zychlinsky A., Moreno E. MyD88, but not toll-like receptors 4 and 2, is required for efficient clearance of Brucella abortus. - Infect Immun, 73, 5137-5143, 2005. Gupta R.K., Relyveld E.H., Lindblad E.B., Bizzini B., Ben-Efraim S., Gupta C.K. - Adjuvants - a balance between toxicity and adjuvanticity. - Vaccine, 11, 293-306, 1993. Otte J.M., Cario E., Podolsky D.K. - Mechanisms of cross hyporesponsiveness to Toll-like receptor bacterial ligands in intestinal epithelial cells. - Gastroenterology, 126, 1054-1070, 2004. Ogushi K., Wada A., Niidome T., Mori N., Oishi K., Nagatake T., Takahashi A., Asakura H., Makino S., Hojo H., Nakahara Y., Ohsaki M., Hatakeyama T., Aoyagi H., Kurazono H., Moss J., Hirayama T. - Salmonella enteritidis FliC (flagella filament protein) induces human beta-defensin-2 mRNA production by Caco-2 cells. - J Biol Chem, 276, 30521-30526, 2001.

45.

46.

47.

48. 49.

50. 51. 52.

53.

54.

55.

56.

J. DRUG DEL. SCI. TECH., 18 (1) 31-39 2008

Robertson J.M., McKenzie N.H., Duncan M., Allen-Vercoe E., Woodward M.J., Flint H.J., Grant G. - Lack of flagella disadvantages Salmonella enterica serovar Enteritidis during the early stages of infection in the rat. - J Med Microbiol, 52, 91-99, 2003. Dalle F., Jouault T., Trinel P.A., Esnault J., Mallet J.M., d'Athis P., Poulain D., Bonnin A. - Beta-1,2- and alpha-1,2-linked oligomannosides mediate adherence of Candida albicans blastospores to human enterocytes in vitro. - Infect Immun, 71, 7061-7068, 2003. Wagner S., Lynch N.J., Walter W., Schwaeble W.J., Loos M. Differential expression of the murine mannose-binding lectins A and C in lymphoid and nonlymphoid organs and tissues. - J Immunol, 170, 1462-1465, 2003. Salman H.H., Gamazo C., Campanero M.A., Irache J.M. Salmonella-like bioadhesive nanoparticles. - J Control Release, 106, 1-13, 2005. Cronkhite R.I., Michael J.G. - Sub-compartmentalization of the gastrointestinal (GI) immune system determined with microbeads that differ in release properties. - Vaccine, 22, 2106-2115, 2004. McSorley S.J., Ehst B.D., Yu Y., T.. G.A. - Bacterial flagellin is an effective adjuvant for CD4 T cells in vivo. - J Immunol., 169, 3914, 2002. Honko A.N., Mizel S.B. - Effects of flagellin on innate and adaptive immunity. - Immunol Res, 33, 83-102, 2005. Umamaheswari R.B., Jain S., Tripathi P.K., Agrawal G.P., Jain N.K. - Floating-bioadhesive microspheres containing acetohydroxamic acid for clearance of Helicobacter pylori. - Drug Deliv, 9, 223-231, 2002. Hattori Y., Kawakami S., Suzuki S., Yamashita F., Hashida M. - Enhancement of immune responses by DNA vaccination through targeted gene delivery using mannosylated cationic liposome formulations following intravenous administration in mice. - Biochem Biophys Res Commun, 317, 992-999, 2004. Dalle F., Dumont L., Franco N., Mesmacque D., Caillot D., Bonnin P., Moiroux C., Vagner O., Cuisenier B., Lizard S., Bonnin A. - Genotyping of Candida albicans oral strains from healthy individuals by polymorphic microsatellite locus analysis. - J Clin Microbiol, 41, 2203-2205, 2003. Engering A.J., Cella M., Fluitsma D., Brockhaus M., Hoefsmit E.C., Lanzavecchia A., Pieters J. - The mannose receptor functions as a high capacity and broad specificity antigen receptor in human dendritic cells. - Eur J Immunol, 27, 2417-2425, 1997. Jain S., Singh P., Mishra V., Vyas S.P. - Mannosylated niosomes as adjuvant-carrier system for oral genetic immunization against Hepatitis B. - Immunol Lett, 101, 41-49, 2005.

Acknowledgements This work was supported by grants from the “Ministerio de Educación y Ciencia de España, CICYT” (Projects SAF 2001-0690-C03 and AGL2004-07088-CO3-02/GAN), “Agencia Española de Cooperacion Internacional, AECI”, Foundations “Universitaria de Navarra” and “María Francisca de Roviralta”, and “Asociación de Amigos Universidad de Navarra” in Spain.

Manuscript Received 10 September 2007, accepted for publication 9 November 2007.

39