Design of biodegradable particles for protein delivery

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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.

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 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], [2], [3]. In the 1980s, it was widely accepted that the major route for the transport of particles across the intestinal mucosa was their 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 μm) 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], [8], [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.

Section snippets

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%)

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

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.

Acknowledgments

This work was financed by a grant from the Commission of Science and Technology (CICYT-SAF 97-0169) and Pierre Fabre Ibérica. 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 Ferreirós and Professor Florencio Martı́nez, of the University of Santiago de Compostela, for their advice.

References (28)

  • J. Iida et al.

    Stimulation of non-specific hosts resistance against Sendai virus and Escherichia coli infections by chitin derivatives in mice

    Vaccine

    (1987)
  • K. Nishimura et al.

    Adjuvant activity of chitin and its derivatives

    Vaccine

    (1984)
  • K. Nishimura et al.

    Adjuvant activity of chitin derivates in mice and guinea-pigs

    Vaccine

    (1985)
  • A. Almeida et al.

    Immune response to nasal delivery of antigenically intact tetanus toxoid associated with poly(l-lactic acid) microspheres in rats, rabbits and guinea-pigs

    J. Pharm. Pharmacol.

    (1993)
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