Design of biodegradable particles for protein delivery

Journal of Controlled Release 78 (2002) 15–24 www.elsevier.com / locate / jconrel

Design of biodegradable particles for protein delivery ´ ´ P. Calvo, M.J. Alonso* A. Vila, A. Sanchez, M. Tobıo, Department Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain Received 20 February 2001; accepted 4 June 2001

Abstract Major research issues in protein delivery include the stabilization of proteins in delivery devices and the design of appropriate protein carriers in order to overcome mucosal barriers. We have attempted to combine both issues through the conception of new biodegradable polymer nanoparticles: (i) poly(ethylene glycol) (PEG)-coated poly(lactic acid) (PLA) nanoparticles, chitosan (CS)-coated poly(lactic acid–glycolic acid (PLGA) nanoparticles and chitosan (CS) nanoparticles. These nanoparticles have been tested for their ability to load proteins, to deliver them in an active form, and to transport them across the nasal and intestinal mucosae. Additionally, the stability of some of these nanoparticles in simulated physiological fluids has been studied. Results showed that the PEG coating improves the stability of PLA nanoparticles in the gastrointestinal fluids and helps the transport of the encapsulated protein, tetanus toxoid, across the intestinal and nasal mucosae. Furthermore, intranasal administration of these nanoparticles provided high and long-lasting immune responses. On the other hand, the coating of PLGA nanoparticles with the mucoadhesive polymer CS improved the stability of the particles in the presence of lysozyme and enhanced the nasal transport of the encapsulated tetanus toxoid. Finally, nanoparticles made solely of CS were also stable upon incubation with lysozyme. Moreover, these particles were very efficient in improving the nasal absorption of insulin as well as the local and systemic immune responses to tetanus toxoid, following intranasal administration. In summary, these results show that a rational modification in the composition and structure of the nanoparticles, using safe materials, increases the prospects of their usefulness for mucosal protein delivery and transport.  2002 Elsevier Science B.V. All rights reserved. Keywords: Nanoparticles; Protein carriers; Vaccine carriers; Chitosan; Poly(lactic acid)–poly(ethylene glycol); Nasal administration; Oral administration

1. Introduction Significant advances in biotechnology have resulted in the discovery of a large number of therapeutic and antigenic proteins. However, the problem to be faced at present is the development of suitable *Corresponding author. Tel.: 134-981-594-488, ext. 14885; fax: 134-981-547-148. E-mail address: [email protected] (M.J. Alonso).

protein delivery devices. Important efforts have already been focused on the design of carriers for the transport of proteins across mucosal barriers, i.e. nasal and intestinal mucosae. Among them, polymeric nanoparticles and microspheres have shown a certain degree of success for the delivery of proteins and vaccines to the systemic circulation and to the immune system [1–3]. In the 1980s, it was widely accepted that the major route for the transport of particles across the intestinal mucosa was their

0168-3659 / 02 / $ – see front matter  2002 Elsevier Science B.V. All rights reserved. PII: S0168-3659( 01 )00486-2

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uptake by the M cells which overlie the lymphoid tissue [4]. At that time, the observation that this transport was particularly intense for hydrophobic microparticles (smaller than 10 mm) became quite popular [5]. However, the information accumulated in the last years has emphasized the importance of the size and revealed the advantages of the nanoparticles over the microspheres [6]. More specifically, some investigators have observed that the number of nanoparticles which cross the intestinal epithelium is greater than the number of microspheres, and that not only the M cells but also the normal enterocytes are involved in the transport [7–9]. Despite the progress of the knowledge in this field, present limitations of nanoparticles as transmucosal protein delivery systems include their instability [10] in contact with the gastrointestinal fluids and their limited interaction and transport across mucosal barriers [11]. Additionally, physiological factors affecting nanoparticulate absorption and their interdependence with the physicochemical properties of the polymeric carrier are not yet well understood [9]. Nevertheless, there are important biopharmaceutical and technological principles that may be taken into account for the design of appropriate protein carriers for mucosal administration. With these principles in mind, over the last few years we have designed new types of nanoparticulate systems intended to improve the transport of proteins associated to them, following either nasal or oral administration. These systems are: (i) PEG-coated PLA nanoparticles, (ii) CS-coated PLGA nanoparticles and (iii) CS nanoparticles. The PEG coating around PLA nanoparticles was conceived with the intention of making these nanoparticles more stable when in contact with physiological fluids. The idea behind this was that the PEG brush would hinder protein / enzyme adsorption, thereby avoiding the harsh environment to which the particles are exposed until they reach the absorbing epithelium. CS was also selected as a coating material for PLGA nanoparticles because of its recognized mucoadhesivity, biodegradability and ability to enhance the penetration of large molecules across mucosal surfaces [12]. Hence, this coating was expected to improve the interaction of the hydrophobic PLGA nanoparticles with the nasal and intestinal absorbing epithelia. Besides, the idea of

making particles consisting solely of hydrophilic polymers, i.e. CS, became appealing to us in order to avoid the use of organic solvents and high energy sources required for the formation of hydrophobic nanoparticles. Therefore, the major goals of the work presented here have been to create new biodegradable nanoparticles appropriately tailored for the incorporation of proteins, and to evaluate their potential as protein carriers for either oral or nasal administration. With these objectives in mind, we have selected low and high molecular weight proteins such as insulin (MW 5800 Da) and tetanus toxoid (TT) (MW 150 000 Da) as model compounds.

2. Materials and methods

2.1. Chemicals and animals The materials and methods used for the preparation and evaluation of PEG–PLA nanoparticles containing TT have been described in Refs. [13,14]. Those used for the preparation and evaluation of CS nanoparticles containing insulin can also be found in Refs. [15,16]. The chemicals and animals used for the preparation and evaluation of CS-coated PLGA nanoparticles and CS particles are described below. CS in the form of hydrochloride salt (Protasan  110 Cl, Mn . 50 kDa, deacetylation degree: 87%) was purchased from Pronova Biopolymer (Norway). Pentasodium tripolyphospate (TPP), poly(vinylalcohol) (PVA) and trehalose were supplied by Sigma (USA). Glucose was provided by Merck (Darmstadt, Germany). Ultrapure water (Milli-Q Plus, Millipore Iberica, Spain) was used throughout. Purified TT (MW 150 000 Da, 85–95% monomeric) dissolved in phosphate buffer saline, pH 7.4, was kindly donated by the World Health Organization (WHO). Antitetanus monoclonal antibody, purified guinea-pig antitetanus immunoglobulin G (IgG) and mouse antitetanus immunoglobulin G standard were obtained from the National Institute for Biological Standards and Control (NIBSC) (Hertfordshire, UK), rabbit antiguinea pig IgG peroxidase conjugate, goat antimouse IgG peroxidase conjugate, goat antimouse IgA peroxidase conjugate and the enzyme substrate

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(ABTS), pilocarpine and pentobarbital were purchased from Sigma (Madrid, Spain). Male BALB / c mice (6 weeks old, 22–25 g), from the Central Animal House of the University of Santiago de Compostela (Spain), were used. They were kept in a 12 h light–dark cycle and at a temperature of 20628C. The animals were allowed access to food and water ad libitum.

2.2. Preparation and characterization of nanoparticles The preparation conditions of PLA and PEGcoated PLA nanoparticles are described elsewhere [13,14]. CS-coated PLGA nanoparticles containing TT with a trace of 125 I-TT were obtained using a critically modified double emulsion process: lecithin was incorporated into the organic phase containing the PLGA and CS was dissolved in the external aqueous phase [17]. More specifically, 50 ml of the TT solution, containing 500 mg of the toxoid, were emulsified in a 1 ml solution of PLGA (50 mg) and lecithin (2 mg) in ethyl acetate, by sonication for 15 s (20 W) (Sonifier 250, Branson). Then, 2 ml of an aqueous PVA solution (1% w / v) containing CS (0.2%) were added to this emulsion and the resulting emulsion was sonicated for 15 s (20 W). The double emulsion was diluted in 100 ml of a PVA solution (0.3%, w / v) and the solvent was rapidly eliminated by evaporation under vacuum. Finally, the nanoparticles were isolated by centrifugation at 22 000 g for 30 min (AvantiE 30, Beckman, Spain) and washed three times with water. CS nanoparticles containing TT were prepared according to the procedure previously developed by our group [18,19], based on the ionotropic gelation of CS with TPP anions. More specifically, the particles formed spontaneously upon addition of 1.2 ml of an aqueous TPP solution (0.84 mg / ml) to 3 ml of CS solution (2 mg / ml, 6 mg chitosan) under magnetic stirring. TT was incorporated into the TPP solution (600 mg of antigen). Nanoparticles were isolated by centrifugation at 16 000 g on a glucose bed, for 40 min. Supernatants were discarded and nanoparticles were resuspended in 5% glucose for their administration. The characterization of the particles, in terms of

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particle size distribution, zeta potential and encapsulation efficiency was performed as described elsewhere [13,16,19].

2.3. Stability of nanoparticles in the presence of lysozyme The stability of the nanoparticles was analyzed following their incubation at 378C in a solution of lysozyme in purified water (1 mg / ml) under moderate stirring. The physicochemical properties of the nanoparticles (mean particle size and zeta potential) were monitored during the incubation process, as previously described [13,16,19].

2.4. Absorption and biodistribution of TT-loaded PEG–PLA nanoparticles and CS-coated PLGA nanoparticles after oral and nasal administration The experimental conditions for the nasal and oral administration of PEG–PLA nanoparticles containing 125 I-TT to rats and the further determination of 125 I-TT are described elsewhere [13,14]. The conditions in which absorption and biodistribution of 125 I-TT loaded into CS-coated PLGA nanoparticles were studied are those previously indicated for PEG– PLA nanoparticles [13].

2.5. Immune response to TT associated to CS nanoparticles The immunogenicity of TT containing nanoparticles was assessed in BALB / c mice following intranasal immunization. A 10-mg amount of the antigen (associated with 70 mg of CS) were given in 20 ml of saline solution (10 ml into each nostril) on days 1, 8 and 15. All animals were conscious during the administration. Blood samples were taken from animals at weeks 2, 4, 12, 18 and 24 postadministration. The serum samples were maintained at 2208C prior to analysis. IgG antibody levels in serum were determined using an ELISA test. Firstly, TT (4 ml / ml) in carbonate buffer (pH 9.6) was added to microplates (Corning, NY, USA) and incubated overnight at 48C in a humid container. The wells were washed three times with PBS containing 0.05% (w / v) Tween  20 (PBST). To minimize non-specific interactions, 100

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ml of PBST containing 2.5% (w / v) of dried skimmed milk powder (PBSTM buffer) were added to the wells, and then incubated for 1 h at 378C in a humid container. After washing the plate three times with PBST, a reference preparation (in the case of evaluation of IgG (9 I.U. / ml)) and samples serially diluted in PBSTM were added. The plates were incubated at 378C for 2 h in a humid container and washed. Then, 100 ml of antimouse IgG peroxidase conjugate diluted 1:2000 in PBSTM was added and incubated for another 1 h at 378C. The plates were washed and the substrate (ABTS) 0.5 mg / ml in 0.05 M citric acid, pH 4.0, was added. Following color development (30 min) plates were read at 405 nm on an microplate reader (3550-UV, Bio-Rad, Spain).

3. Results and discussion Our research in the field of protein delivery has focused on the idea of devising nanoparticles for the transport of proteins across mucosal surfaces. For the rational design of these particles we have taken into account some biological considerations, i.e. the presence of proteins and enzymes in the mucus and physiological fluids. With this idea in mind, we chose three different safe polymers to make these nanoparticles, PEG–PLA, PLGA and CS, and developed or adapted nanoencapsulation techniques in order to change the surface composition and to load proteins into these particles. As indicated in the Introduction, PEG was chosen with the intention of making the hydrophobic PLA or PLGA nanoparticles more stable in physiological fluids, whereas the idea of using CS was based on its mucoadhesive and permeability enhancing properties. The relevant

characteristics of the nanoparticles which were selected for in vivo studies, such as size, zeta potential and protein loading capacity, are presented in Table 1. The impact that the specific design of these nanoparticles had on their in vivo behavior as mucosal carriers for protein administration is described below.

3.1. PEG-coated PLA nanoparticles The conditions under which these particles were prepared were conveniently adapted in order to encapsulate proteins and provide a controlled protein release. The size of these nanoparticles may easily be adjusted, from 100 to 1000 nm, by modifying the formulation parameters. As seen in Table 1, those selected for the in vivo studies had a mean size of 196 nm. The zeta potential has a negative value, lower than that of the particles without a PEG coating. These particles were loaded with TT, which was chosen as a model of a large protein (150 000 Da). The encapsulation efficiency was only of 33% due to the partition of the protein between the inner and external aqueous phases. However, the in vitro studies showed that these nanoparticles release antigenically active TT in a pulsatile manner [13]. These positive data in terms of the activity of the protein released over the time led us to adduce the hypothesis that the presence of PEG might improve the stability of the protein encapsulated in the PLA nanomatrix. Additionally, in vitro results describing the stability of the nanoparticles in the gastrointestinal fluids revealed that the PEG coating has a role in preventing the enzyme mediated aggregation typically observed for PLA nanoparticles [14]. More recently, we studied the effect of lysozyme on the stability

Table 1 Particle size, zeta potential, theoretical loading and encapsulation efficiency values of CS, PEG–PLA and CS–PLGA nanoparticles containing TT and CS nanoparticles containing insulin Polymer

Protein loaded

Size (nm)

z Potential (mV)

Theoretical loading (%)

Encapsulation efficiency (%)

PLA PEG–PLA CS–PLGA CS CS

Tetanus toxoid Tetanus toxoid Tetanus toxoid Tetanus toxoid Insulin

192612 196620 500629 354627 337614

247.961.5 223.961.2 121.861.1 137.165.9 136.960.3

1 1 1 10 40

36.760.3 31.160.5 90.063.8 55.163.4 94.762.1

Results are presented as mean (n53)6standard deviation.

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of the particles and found that while PLA nanoparticles were stable in the absence of the enzyme, they aggregated rapidly upon incubation with lysozyme. This aggregation was concomitant with an inversion of the zeta potential values. Conversely, the nanoparticles having a PEG coating remained stable upon incubation with lysozyme and suffered only a neutralization of their surface charge (Fig. 1a and b). Consequently, it could be inferred that the PEG coating hinders the interaction with the lysozyme. The potential of these nanoparticles as protein carriers for nasal and oral administration was further investigated using 125 I-radiolabelled TT [13,14].

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Analysis of radioactivity in blood, lymph nodes and other relevant tissues indicated that PEG-coated nanoparticles were able to enhance the transport of TT across the nasal and the intestinal epithelium. More specifically, the amounts of radioactivity recovered in the blood circulation at 1 h post administration were 10- and 5-fold those observed for PLA nanoparticles, following either nasal or oral administration, respectively (Fig. 2a and b). Furthermore, this concentration remained fairly constant for at least 24 h. On the other hand, the percentage of radioactive antigen that reached the lymph nodes was also significantly greater for the PEG-coated

Fig. 1. Particle size (a) and zeta potential (b) values of PLA, PEG–PLA, CS–PLGA and CS nanoparticles before and after incubation in the presence of lysozyme.

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Fig. 3. Percentages of 125 I-TT recovered per g of tissue with respect to the original dose in lymph nodes following intranasal and oral administration of 125 I-TT-loaded PLA and 125 I-TT-loaded PEG–PLA nanoparticles (mean6S.D., n54–8).

Fig. 2. Percentages of 125 I-TT recovered per gram of blood with respect to the original dose following intranasal (a) and oral (b) administration of 125 I-TT-loaded PLA and 125 I-TT-loaded PEG– PLA nanoparticles (mean6S.D., n54–8).

nanoparticles than for the uncoated ones, following either intranasal or oral administration (P.0.05) (Fig. 3). It was also interesting to observe, when comparing both modalities of administration, that the percentage of radioactivity associated with PEG– PLA nanoparticles that reached the blood circulation was greater for the nasal route than for the oral route. In contrast, the corresponding percentage in lymph nodes was greater following oral rather than nasal administration. This could be related to the different biological fluids, permeability of mucosal epithelia and extension of the lymphoid tissue associated with these mucosae. Irrespective of the differences ob-

served for both modalities of administration, the most significant conclusion that could be drawn from these data is that the presence of a PEG coating around the particles leads to a greater absorption of the encapsulated material. Whether this is due solely to the improved stability of the particles or whether it is also related to an enhanced transport of the nanoparticles should be further elucidated. Nevertheless, the preliminary results of the transport of these particles through Caco-2 cells suggest that the PEG coating plays a role in facilitating this transport (results not shown). Furthermore, the favorable transport of PEG–PLA nanoparticles has recently been corroborated by the increasing and long-lasting immunogenic response elicited following intranasal administration of TT-loaded PEG–PLA nanoparticles [article in preparation]. These results agree with those obtained by Jung et al. [9] for comb PLGA polymers. These authors observed that the presence of hydrophilic polymers (polysulphobutylvinyl alcohol) attached to the PLGA backbone provided enhanced affinity of the nanoparticles for the Caco-2 cells. The greater permeability of these novel particles would also explain the greater response elicited by TT associated with them, following oral administration [9].

3.2. CS-coated PLGA nanoparticles These nanoparticles were also designed with the idea of facilitating the interaction of the nanoparti-

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cles with the mucosal surfaces. These nanoparticles can easily be obtained by nanoprecipitation or solvent evaporation techniques by introducing a simple but critical modification in the formation process: CS is dissolved in the external aqueous medium in which the formation of the particles takes places whereas lecithin is introduced in the organic phase in which the polymer is dissolved. Using this approach, CS molecules anchor to the surface of the particles because of the physical entanglement between polymer chains that is further facilitated by the ionic interaction between the negatively charged surfactant (lecithin) and the positively charged CS molecules [17]. The presence of CS around the particles was observed by transmission electron microscopy and reflected by an increase in the hydrodynamic size and by the positive zeta potential of the nanoparticles (Table 1). Results in Table 1also show the significant increase in the TT encapsulation efficiency for the nanoparticles coated with CS as compared to the values obtained for PLA or PEG–PLA nanoparticles. Additionally, the CS coating was found to protect these particles from aggregation upon incubation with lysozyme (Fig. 1a). From these data we could conclude that the positive charge of CS limits the adsorption of lysozyme (also positively charged) and, hence, the enzyme-mediated aggregation observed for PLA particles. These results agree with those reported by Calvo et al. [20] who observed the positive effect of a CS coating in preventing the typical aggregation of nanocapsules caused by lysozyme. Interestingly, following incubation with lysozyme the zeta potential of these particles changed from positive values to values close to neutrality (Fig. 1b), while in the absence of lysozyme the zeta potential was constant. This observation suggests that lysozyme interacts with CS and changes its organization at the surface of the nanoparticles. It can also be speculated that the degradation of the CS coating is accelerated by lysozyme [21]. However, the surface charge reduction was observed immediately upon addition of lysozyme at room temperature. This rapid change in the surface properties discards, consequently, the possibility of partial loss of the coating due to the polymer enzymatic degradation. CS was chosen as a coating polymer because of its recognized mucoadhesive and permeability enhanc-

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ing properties [12]. Consequently, CS presence on the surface was supposed to increase the interaction of the nanoparticles with the mucus as well as their transport across epithelia. The role of CS in improving the transport of proteins across mucosal surfaces was investigated following intranasal administration of 125 I-radiolabelled TT. The radioactivity levels in the blood circulation (Fig. 4) led us to the conclusion that the CS-coated PLGA nanoparticles provided greater protein transport than PLA nanoparticles. This could be attributed to the attachment of the particles to the nasal mucosa, facilitated by the CS coating. This hypothesis agrees well with a previous report by Kawashima et al. [22] which shows the adsorption of CS-coated nanoparticles to the rat everted intestinal sac. On the other hand, the comparison of the results presented in Fig. 2a and Fig. 4 leads to the conclusion that the PEG coating was more efficient than the CS coating in facilitating the transport of the associated antigen. A possible explanation of this behavior could be found in the different interaction mechanisms of the nanoparticles with the nasal mucosa. In fact, whereas PEG–PLA nanoparticles do not interact with the mucine, those coated with CS are supposed to stick to the mucus layer. Moreover, we also know that PEG–PLA nanoparticles cross the Caco-2 cells by a transcellular pathway [results not shown], however, the mechanisms of interaction and transport of CS-coated particles remains to be investigated.

Fig. 4. Percentages of 125 I-TT recovered per gram of blood with respect to the original dose following intranasal administration of 125 I-TT-loaded CS–PLGA and 125 I-TT-loaded PLA nanoparticles (mean6S.D., n54–8).

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3.3. CS nanoparticles Being conscious of the harmful conditions required for the preparation of hydrophobic PLA and related nanoparticles (organic solvent–water interfaces), we designed, as an alternative, nanoparticles made solely of hydrophilic polymers, i.e. CS nanoparticles [18]. We obtained these nanoparticles through a spontaneous ionic gelation process, in an aqueous medium, in the presence of a counter anion such as sodium tripolyphosphate. Different types of CS (different molecular weights, type of salt and deacetylation degree) as well as PEG–CS can be used for the formation of these particles. Besides, other hydrophilic polymers, e.g. poloxamer, may be very easily incorporated within the nanoparticles. The size and zeta potential of these particles can be conveniently adjusted. Those selected for the in vivo studies are in the 300–400 nm range and have a positive zeta potential (Table 1). A very interesting feature of these novel particles is their great protein loading capacity. Insulin, used as a model compound, was incorporated into these particles very efficiently reaching final loading values up to 50% (mg insulin / 100 mg CS). These particles were also found to be stable upon their incubation in the presence of lysozyme. As shown in Fig. 1a and b, only a minor reduction in size was observed whereas the surface charge remains unmodified. The slight size reduction could be attributed to the detachment of some CS fragments caused by a minor polymer degradation [21]. The utility of these nanoparticles for nasal protein delivery was first investigated in conscious rabbits, using insulin as a model compound. Results of the plasma glucose levels following intranasal instillation of insulin-loaded nanoparticles showed their ability to enhance insulin absorption. Furthermore, glucose levels obtained for CS nanoparticles were significantly lower than those for CS solutions, thus indicating that CS nanoparticles are more efficient than CS solutions for delivering insulin to the blood circulation [15,16]. It is still not clear how CS nanoparticles enhance the transport of insulin. However, considering the dose-dependent decrease in the transepithelial resistance of the Caco-2 cell monolayers caused by CS [23] we assume that the nanoparticles adhere to mucus, providing a high

concentration of CS to the underlying epithelium while simultaneously delivering the associated protein. Nevertheless, further studies are required in order to fully understand how the interaction between these particles and the nasal epithelial cells occurs. More recent studies, aimed at evaluating the performance of these nanoparticles for immunization, provided additional evidence of their potential for nasal protein delivery. TT-loaded CS nanoparticles were administered by intranasal instillation to conscious mice and the systemic and mucosal immune responses were monitored. Fig. 5 shows that the antitetanus IgG levels elicited by the TT-loaded nanoparticles were significantly higher than those corresponding to the fluid vaccine. This trend is clear evidence for the adjuvant effect of the nanoparticles. Another remarkable observation was that these differences increased with time. Similar conclusions could be drawn from the antitetanus IgA titers detected in saliva, bronchoalveolar and intestinal lavages at 6 months postadministration (results not shown). The mechanism by which CS nanoparticles enhance serum and secretory immune responses to mucosally applied macromolecules is presently under investigation. However, as in the case of insulin, we assumed that CS facilitates the access of the antigen to the nasal-associated lymphoid tissue (NALT) as well as to the antigen presenting cells (APC) underlying the nasal epithelium. This has actually been a hypothesis postulated to explain the enhanced

Fig. 5. IgG antibody levels following intranasal administration of TT entrapped in CS nanoparticles (70 kDa) or in PBS solution to mice (mean6S.E.M., n56–9).

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humoral and mucosal responses to protein antigens administered intranasally in the form of a CS solution [24,25]. Nevertheless, the large size of the antigen (MW TT: 150 000 Da) and the long-lasting and increasing immune response observed in this study suggest that other mechanisms, different to the merely increased paracellular transport of the antigen, might be responsible for the positive behavior of CS nanoparticles. Indeed, information regarding the immunostimulating properties of CS administered either nasally [26] or parenterally [27,28] led us to accept the possibility that some CS molecules released at the mucosal surface, or even the whole particles, might be able to cross the epithelium and elicit the inherent CS immunostimulatory properties. The increasing and long lasting responses would also support the hypothesis that the nanoparticles may cross the nasal mucosa, thereby transporting the associated antigen and deliver it in a sustained form, for extended periods of time.

4. Conclusions The work presented led us to the conclusion that although general statements on the potential of nanoparticles as carriers for mucosal protein delivery cannot be made, their rational design can open new optimistic prospects in this area. More specifically, hydrophobic particles coated with hydrophilic polymers such as PEG or CS or particles made solely of hydrophilic polymers, i.e. CS, have shown an improved ability to deliver proteins and vaccines across the nasal and intestinal mucosae. Experiments are in progress in our laboratory to further investigate the mechanism of action of these particles.

Acknowledgements This work was financed by a grant from the Commission of Science and Technology (CICYT´ SAF 97-0169) and Pierre Fabre Iberica. The authors wish to thank the WHO and the NIBSC for the donation of TT and ELISA reagents, and to thank ´ Professor Teresa Criado, Professor Carlos Ferreiros ´ and Professor Florencio Martınez, of the University of Santiago de Compostela, for their advice.

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