Binding and uptake of biodegradable poly-dl-lactide micro- and nanoparticles in intestinal epithelia

Abstract

The use of biodegradable particles as oral delivery vehicles for macromolecular drugs was investigated. We evaluated the binding, uptake and absorption of poly-dl-lactide (PLA) micro- and nanoparticles in Caco-2 monolayers and in ileal tissue and gut associated lymphoid tissue (GALT) of anaesthetised rats and rabbits. Using a range of experimental techniques, we found that approximately 10% of administered micro- and nanoparticles were adsorbed to the apical membranes of each of the five intestinal models. Nanoparticles were found to be absorbed better than microparticles. Overall, little discrimination in uptake patterns was evident between Peyer's patch (PP) and non-PP tissue while rat ileum showed a greater uptake capacity than rabbit. Our results show that uptake of PLA particles was low capacity, size-dependent and predominantly transcellular in all systems. A low proportion of the apically-bound particles was absorbed, with uptake exclusion evident for particles >4μm. The affinity of PLA particles for intestinal epithelia and GALT needs to be greatly enhanced in order to achieve improved oral bioavailability of macromolecules.

Introduction

Biodegradable micro- and nanoparticles are being widely investigated as systems to deliver poorly absorbed drugs including peptides, proteins and vaccines to mucosal surfaces (see Couvreur and Puisieux, 1993, Wilding et al., 1994, for reviews). Oral administration of encapsulated particles can protect labile macromolecules from stomach acid and from digestion in the gastrointestinal tract. However, micro- and nanoparticles have been found to be poorly absorbed by the intestine with only a small percentage of particles appearing in lymph nodes, blood, liver and spleen (Nefzger et al., 1984). Nonetheless, drug-loading of particles has been reported to enhance or prolong the peroral bioavailability of a number of drugs, including vincamine (Maincent et al., 1986), hydrocortisone (Alpar et al., 1990), heparin (Brady and Ramtoola, 1996), insulin and dicumarol (Mathiowitz et al., 1997). In addition to the potential for enhancing drug bioavailability via particle uptake mechanisms, particulate oral delivery systems also have slower transit times than larger dosage forms and can increase the local concentration gradient across absorptive cells, thereby enhancing local and systemic delivery of both free and bound drugs across the gut (Kreuter et al., 1989).

However, results have been conflicting with regard to time-dependent particle uptake. Alpar et al. (1989), reported that 1 μm diameter latex particles were detected in blood within 10 min of oral administration, with maximal adsorption observed after 45 min. Pappo and Ermak (1989)found that 90 min was required for 670 nm latex particles to translocate to the domes of rabbit Peyer's patch (PP). Eldridge et al. (1990), showed that uptake of 1 to 10 μm poly(dl- lactide-co-glycolide (PLGA) particles increased up to day 4 following a single oral dose and that particles of <5μm were transported through the lymphatic system, while larger particles were retained in PPs. Values quoted for percentage absorption of nanoparticles have ranged from 1 to 5% (Kukan et al., 1989and Le Ray et al., 1994, respectively), differences arising from the selected polymeric material or from the intestinal model and species selected.

Consensus has not been reached on the mechanisms of particle uptake by intestinal epithelia. Most evidence suggests that the favoured site for uptake is the PP lympho-epithelial M cell (Lefevre et al., 1985, Eldridge et al., 1990, Jani et al., 1992). However, paracellular transport of particles has been favoured by others (Volkheimer and Schulz, 1968, Aprahamian et al., 1987) while there is also evidence for particle endocytosis by intestinal enterocytes (Sanders and Ashworth, 1961, Kreuter et al., 1989). Indeed, it was recently reported that particles in the size range 40 to 120 nm were translocated both transcellularly and paracellularly (Mathiowitz et al., 1997).

The aim of this study was to examine the interactions of poly-dl-lactic acid (PLA) particles with Caco-2 monolayers and with intestinal PP and non-PP ileal tissue loops of rat and rabbit gut in situ in order to investigate the binding, uptake and mechanism of absorption / translocation of the particles using several complimentary methodologies. PLA particles were also loaded with either fluorescein or with ferritin to serve as fluorescent and EM markers respectively. Two different size ranges of particles were investigated, broadly termed microparticles (2 to 30 μm) and nanoparticles (300 to 2000 nm). Overall, our results showed that while micro- and nanoparticles adsorbed equally to the apical membranes of all the tissues tested, only particles <4μm went on to be absorbed by a predominantly transcellular process in each model.