PLA-PEG particles as nasal protein carriers: the influence of the particle size
Introduction
At the beginning of the nineties, Gref et al. (1994) presented PLA-PEG nanoparticles (NP) as long circulating systems for intravenous administration. This initial work has been followed by a number of reports that have shown the potential of these new carriers as controlled release systems for parenteral administration (Peracchia et al., 1999, Verrechia et al., 1995). Over the last years, we have attempted to explore the potential of these systems as transmucosal carriers for proteins (Tobío et al., 1998, Tobío et al., 2000). With this idea in mind, we encapsulated radiolabelled TT into PLA and PLA-PEG NP and evaluated the TT absorption following nasal and oral administration to rats. The results showed that, irrespective of the administration route, the TT levels in the blood stream and lymph nodes were significantly higher for PLA-PEG NP than for PLA NP. Consequently, these initial studies suggested the potential of PLA-PEG NP as transmucosal protein delivery systems. More recent studies aimed at evaluating the performance of these NP for immunization provided additional evidence of their potential for nasal protein delivery (Vila et al., 2002a). More specifically, the anti-tetanus IgG levels elicited following nasal administration of TT-loaded NP were significantly higher than those corresponding to the fluid vaccine. Nevertheless, despite these positive results, the mechanism of action of these NP at the nasal mucosa level and the specific role of the PEG coating has not been clearly identified yet. In this sense, we should keep in mind some interesting features of PEG, with respect to its application for mucosal drug administration. These are related to its mucoadhesion promoting effect (Ascentiis et al., 1995, Peppas, 1998) and its ability to increase the stability of drugs on the nasal mucosa (Bechgaard et al., 1999; Lindhardt et al., 2000).
Besides the potential benefit of the PEG coating, the idea of using nanoparticles as nasal drug carriers was supported by previous work that showed the transport of model polystyrene particles across the nasal mucosa. Indeed, Almeida et al. (1993) investigated the transport of fluorescently labelled, carboxylated polystyrene NP from the nasal cavity into the systemic circulation. The authors observed that the NP crossed the nasal barrier and reached the blood stream. Some years later, Huang and Donovan (1996) reported the effect of size on the particulate uptake through a rabbit nasal mucosa mounted in a diffusion chamber. Following these initial reports and also the results of our own work (Tobío et al., 1998, Tobío et al., 2000), other authors have investigated further the influence of the size on the nasal transport of nanoparticles. For example, Brooking et al. (2001) studied the transport of 125I-radiolabelled latex NP across the nasal mucosa of rats using a range of particle sizes (20, 100, 500 and 1000 nm). They found a relationship between the intensity of the transport and the particle size. In addition to these transport studies using model polystyrene particles, other authors observed that the size of PLA particles influences the immune responses to nanoencapsulated antigens following nasal administration. Indeed, Somavarapu et al. (1998) showed that the immune response to encapsulated ovalbumin administered intranasally was significantly greater for PLA NP than for PLA microparticles (MP). More recently, Jung et al. (2001) investigated the influence of the size of sulphobutylated poly(vinyl alcohol)-graf-poly(lactide-co-glycolide) particles on the immune response to TT adsorbed onto the particles, administered by the nasal route. The authors found that the induction of antibody responses was influenced by the size of the particles, being the response most important for the antigen associated to the smallest particles.
Taking this previous information on the nasal transport of nanoparticles into account, as well as the results of our own work on the efficiency of PLA-PEG particles as transmucosal antigen carriers, the main purposes of the present work were: first, to study the potential effect of blank PLA-PEG nanoparticles in the nasal absorption of the free toxoid, and, second, to evaluate the influence of the PLA-PEG particles size on their ability to transport the encapsulated TT. Additionally, given the sensitivity of the nasal mucosa to external agents we studied if the use of anaesthesia in the in vivo experiments affects the transport of TT across the rat mucosa.
Section snippets
Chemicals and animals
For the polymer synthesis, d,l-lactide was purchased from Aldrich (Milwakee, USA), monomethoxypolyethylenglycol (MW: 5000 Da) and stannous octoate were obtained from Sigma Chemical (St. Louis, USA). Purified tetanus toxoid (MW: 150,000 Da, 85–95% monomeric) dissolved in phosphate buffer saline, pH 7.4, was kindly donated by the Massachusetts Biological Laboratories (MBL, Boston, USA). Cholic acid (sodium salt) was purchased from Sigma Chemical (Madrid, Spain) and the solvent ethyl acetate was
Preparation and physicochemical characterization of NP and MP
For the preparation of PLA-PEG particles (NP and MP) we chose the double emulsion technique. This is a versatile technique that allowed us to control the size of the particles while achieving significant protein loadings. The characteristics of the particles such as, size, encapsulation efficiency and final loading are shown in Table 1. The formulation parameters were conveniently adapted in order to produce particles of different critical sizes: 200 nm, 1.5, 5 and 10 μm. The encapsulation
Conclusions
In the light of these results, we can conclude that PLA-PEG NP work as carriers that are able to transport efficiently the encapsulated tetanus toxoid through the nasal mucosa. In addition, it can be concluded that the extent of the absorption of the toxoid encapsulated into the particles was dependent on the size of the particles, being more important for the NP than for the MP.
Acknowledgement
The authors are grateful to Rafael Romero for his help in “in vivo” studies.
References (24)
- et al.
Mucoadhesion of poly(2-hydroxyethyl methacrylate) is improved when linear poly(ethylene oxide) chains are added to the polymer network
J. Control. Release
(1995) - et al.
Protein encapsulation and release from poly(lactide-co-glycolide) microspheres: effect of the protein and polymer properties and of the co-encapsulation of surfactants
Eur. J. Pharm. Biopharm.
(1998) - et al.
Radiolabelled biodegradable microspheres for lung imaging
Eur. J. Pharm. Biopharm.
(2000) - et al.
Liposomes as an immunoadjuvant system for stimulation of mucosal and systemic antibody responses against inactivated measles virus administered intranasally to mice
Vaccine
(1995) - et al.
Mucosal immunoadjuvant activity of liposomes: induction of systemic IgG and secretorey IgA responses in mice by intranasal immunization with an influenza subunit vaccine and coadministered liposomes
Vaccine
(1995) - et al.
The stability and immunogenicity of a protein antigen encapsulated in biodegradable microparticles based on blends of lactide polymers and polyethylene glycol
Vaccine
(1999) - et al.
Intranasal absorption of buprenorphine—in vivo bioavailability study in sheep
Int. J. Pharm.
(2000) - et al.
Stealth PEGylated polycyanoacrylate nanoparticles for intravenous administration and splenic targeting
J. Control. Release
(1999) - et al.
The role of PEG on the stability in digestive fluids and in vivo fate of PEG-PLA nanoparticles following oral administration
Colloid. Surfaces B: Biointerf.
(2000) - et al.
Design of biodegradable particles for protein delivery
J. Control. Release
(2002)