Pharmaceutical Nanotechnology
Adhesion of PLGA or Eudragit®/PLGA nanoparticles to Staphylococcus and Pseudomonas

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Abstract

The aim of present study was to examine whether cationic Eudragit® containing poly(lactide-co-glycolide) (PLGA) nanoparticles can adhere to Pseudomonas aeruginosa and Staphylococcus aureus. In order to prepare fluorescent nanoparticles, fluorescein was covalently coupled to PLGA. Fluorescent PLGA and Eudragit®/PLGA nanoparticles were prepared by w/o/w emulsification solvent evaporation. Particle size and zeta potential of the nanoparticles were measured. Nanoparticles were incubated for a short time with P. aeruginosa and S. aureus followed by measurement of the size of nanoparticles and of P. aeruginosa and S. aureus with and without adherent nanoparticles. Flow cytometric measurements were performed to detect the attachment of particles to microorganisms. Eudragit® containing nanoparticles possessed a positive zeta potential, while PLGA nanoparticles were negatively charged. Following adsorption of Eudragit® containing nanoparticles, a size increase for P. aeruginosa was observed. Flow cytometric analyses confirmed that Eudragit® containing particles showed stronger interactions with the test organisms than PLGA nanoparticles. Adhesion of particles was more pronounced for P. aeruginosa than for S. aureus. Cationic Eudragit® containing nanoparticles showed better adhesion to microorganisms than anionic PLGA nanoparticles, which is probably due to enhanced electrostatic interactions.

Introduction

Topical application is the most popular administration route for ocular drugs. However, one of the major problems is their low bioavailability, due to the different defence mechanisms of the eye (Järvinen et al., 1995). The use of colloidal drug delivery systems such as microparticles or nanoparticles has been proposed to improve the bioavailability (Zimmer and Kreuter, 1995, Le Bourlais et al., 1998). They are frequently made of poly(lactic-co-glycolic acid) (PLGA) because of its biocompatibility and biodegradability. An additional approach to optimise ocular drug delivery is the so-called mucoadhesive concept, based on entrapment of particles in the ocular mucus layer and interaction of bioadhesive polymer chains with mucins. This induces a significant increase in precorneal residence time of the preparation (Ludwig, 2005). As the mucus layer at the eye surface is negatively charged, cationic polymers might interact with it (Baeyens and Gurny, 1997). Various polymers have been examined in order to prepare mucoadhesive nanoparticles. Pignatello et al. (2002) developed nanoparticles made of Eudragit® RL100 with good ocular tolerance, no inflammation or discomfort in the rabbit's eye. Positively charged nanoparticles can also be prepared when Eudragit® RL100 is combined with PLGA (Dillen et al., 2006).

Most bacteria carry a net negative surface charge, thereby promoting adhesion on positively charged materials (Gottenbos et al., 2001, Gottenbos et al., 2003, Dunne, 2002). Stenotrophomonas maltophilia is an exception to this rule, possessing an overall positive surface charge at physiological pH (Jucker et al., 1996). Bacterial cell wall polymers such as teichoic acid (Gram-positive bacteria), lipid A (part of the lipopolysaccharide, Gram-negative bacteria), peptidoglycan, and most of the phospholipids are negatively charged (Caroff and Karibian, 2003, Bos and Tommassen, 2004, Fedtke et al., 2004).

The aim of present research is to examine whether nanoparticles made of PLGA or of a mixture of Eudragit® and PLGA can adhere to Gram-positive and Gram-negative microorganisms. This adhesion could provide a sustained antimicrobial activity of the drug incorporated against the target organisms causing eye infections. In order to enable visualisation of nanoparticle adhesion to microorganisms during flow cytometric experiments, fluorescent nanoparticles are required. Fluorescent polystyrene particles are used as a model for the uptake of poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles (Tosi et al., 2005). However, polystyrene nanoparticles are more hydrophobic than PLGA nanoparticles (Panyam et al., 2003). A second possibility is the incorporation of a fluorescent dye, like fluorescein or rhodamin (Torché et al., 2000, White and Errington, 2005). Particles labelled with unbound fluorescein rapidly release the fluorescent dye in the medium (Tosi et al., 2005). Derivatisation of PLGA with covalently bound substances, like fluorescein, and consequent formulation of the resulting fluorescent polymer into nanoparticles, would permit adhesion studies.

In an earlier study, nanoparticles consisting of PLGA or a mixture of PLGA and Eudrgit RL 100 were produced and shown to have a drug loading efficiency of 62% and 68%, respectively. DSC measurements showed that the drug is homogeneously dispersed in the matrix in an amorphous state The release of the drug was tested in vitro, and the model that seemed to fit the release curves best was the Higuchi model, with a rate constant kH of 4.45 ± 1.01 (% h−1/2) for PLGA nanoparticles and a kH of 4.28 ± 0.56 (% h−1/2) for PLGA:Eudragit 3:1 (Dillen et al., 2006).

In present study, blank particles without drug as well as particles loaded with ciprofloxacin HCl are prepared to evaluate adhesion. The model drug ciprofloxacin is one of the most commonly used fluoroquinolones in ophthalmology, due to its broad in vivo activity spectrum against both Gram-positive and Gram-negative ocular pathogens (Hwang, 2004). Therefore, the test microorganisms employed in this experiment are Pseudomonas aeruginosa and Staphylococcus aureus, representing the most common ocular pathogens causing human corneal bacterial infections (Moreau et al., 2002).

Section snippets

Materials

Poly(lactide-co-glycolide) (PLGA) with a lactide:glycolide molar ratio of 52:48 and a molecular weight of 40,000 (Resomer® RG 503 and Resomer® RG 503 H) was obtained from Boehringer Ingelheim (Ingelheim am Rhein, Germany). Resomer RG® 503 H differs from Resomer RG® 503 in that it has free carboxylic end groups in the polymer chain (with an acid number > 3 mg KOH/g). Poly (vinylalcohol) (PVA MW 30,000–70,000) and 5(6)-carboxyfluorescein were purchased from Sigma Chemicals Co. (St. Louis, USA) and

Physicochemical characterisation of nanoparticles

The results of the PCS measurements of average particle size of the different nanoparticle formulations made of PLGA alone or blends of PLGA and Eudragit® RL100 are given in Table 1. Size of PLGA nanoparticles after resuspension in PBS ranged from 230 to 250 nm, while sizes found for Eudragit® containing nanoparticles were around 170–180 nm with a narrow polydispersity index of 0.01–0.08 and 0.04–0.07, respectively. Incorporation of ciprofloxacin HCl did not have an influence on particle size and

Discussion

Fluorescent PLGA and Eudragit®/PLGA nanoparticles were prepared in order to evaluate adhesion to S. aureus and P. aeruginosa. To avoid leakage during experiments, the fluorescent dye fluorescein was covalently coupled to PLGA. Fluorescent particles were prepared by the solvent evaporation method. Polymeric Eudragit® containing particles possessed a small particle size with narrow size distribution and a positive zeta potential. PLGA nanoparticles on the other hand, also possessed a small

Conclusion

This study has shown that treatment of P. aeruginosa with positively charged Eudragit® containing PLGA nanoparticles can cause nanoparticle adhesion to their surface. This adhesion was shown by an increase in size, after incubation with nanoparticles, and confirmed by flow cytometric measurements. An electrostatic interaction between the cationic nanoparticles and negatively charged molecules in the bacterial cell wall is most likely responsible for the adhesion. An adhesion of

Acknowledgements

This research was supported by a grant from the Belgian Fund for Research in Ophthalmology (FRO). K. Dillen is a Research Assistant of the Research Foundation – Flanders (FWO, Belgium). P. Van der Veken and P. Cos are Postdoctoral Fellows of the Research Foundation – Flanders (FWO, Belgium).

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