Sustained ex vivo skin antiseptic activity of chlorhexidine in poly(ϵ-caprolactone) nanocapsule encapsulated form and as a digluconate

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Abstract

In this work, the sustained bactericidal activity of chlorhexidine base loaded poly(ϵ-caprolactone), PCL, nanocapsules against Staphylococcus epidermidis inoculated onto porcine ear skin was investigated. Drug loaded nanocapsules were prepared by the interfacial polymer deposition following solvent displacement method, then characterized by photon correlation spectroscopy, electrophoretic measurements, transmission and scanning electron microscopy. Antimicrobial activity of these colloidal carriers was evaluated (i) in vitro against eight strains of bacteria, and (ii) ex vivo against Staphylococcus epidermidis inoculated for 12 h onto porcine ear skin surface treated for 3 min either with 0.6% chlorhexidine base loaded or unloaded nanocapsules suspended in hydrogel, or 1% chlorhexidine digluconate aqueous solution. Chlorhexidine absorption into the stratum corneum (SC) was evaluated by the tape-stripping method. The results showed that chlorhexidine nanocapsules in aqueous suspension having a 200–300 nm size and a positive charge exhibited similar minimum inhibitory concentrations against several bacteria with chlorhexidine digluconate aqueous solution. Ex vivo, there was a significant reduction in the number of colony forming units (CFUs) from 3-min treated skin with chlorhexidine nanocapsule suspension (5 to <1 log10) compared to chlorhexidine digluconate solution (5 to 2.02 log10) after a 8-h artificial contamination. After a 12-h artificial contamination, both formulations failed to achieve a 5 log10 reduction. Furthermore, from a 3-min treatment with an identical applied dose and a subsequent 12-h artificial contamination, a residual chlorhexidine concentration in the SC was found to be three-fold higher with chlorhexidine nanocapsule suspension than with chlorhexidine digluconate solution. Interestingly, nanocapsules were shown in porcine skin follicles. Consequently, a topical application of chlorhexidine base-loaded positively charged nanocapsules in an aqueous gel achieved a sustained release of bactericide against Staphylococcus epidermidis for at least 8 h. Enhancement of drug delivery by mediating a more direct and prolonged contact between the carrier and (i) bacteria, (ii) skin surface, and (iii) skin follicles was assumed.

Introduction

Nosocomial infections represent the major source of morbidity and mortality for patients in the intensive care unit [1]. Wearing gloves and hand-washing with antiseptics [2], [3] might circumvent transmission of nosocomial pathogens by hands of health care personnel and/or by the bacterial flora of patient’s skin [4]. Various antiseptics including chlorhexidine have been incorporated into hand-washing agents [5]. However, the frequent use of antiseptics and some scrubbing techniques also have been associated with contact dermatitis and allergies [6]. Therefore, the potential of chlorhexidine incorporated either in liposomes or in microspheres to provide controlled delivery of the drug to skin-associated bacteria (e.g. Staphylococcus epidermidis) and sustained activity against Gram-positive and Gram-negative bacteria was investigated [7], [8]. Liposomes, in spite of some advantages over conventional dosage forms, are limited in their use by low encapsulation efficiency, rapid leakage and poor storage stability [9]. Recently, the use of biodegradable polymeric nanoparticles as drug delivery devices has been extensively reviewed [10], and nanocapsules (i.e. nanoparticles having a polymeric shell and core) have been mainly suggested for drug targeting, sustained release for oral and ocular bioavailability improvement purposes [11].

The aim of the present study was to investigate the potential of chlorhexidine base entrapped into poly(ϵ-caprolactone) (i.e. a biodegradable polyester, PCL) nanocapsules [12] to provide sustained bactericidal activity (i) in vitro against several hospital strains of bacteria by determination of minimal inhibitory concentration (MIC), (ii) ex vivo against Staphylococcus epidermidis (i.e. coagulase-negative Staphylococcus associated with the skin responsible for nosocomial infections) applied to porcine ear skin. The topical antimicrobial efficacy of chlorhexidine base loaded PCL nanocapsules was compared to that obtained with a disinfectant-detergent solution of chlorhexidine digluconate. Furthermore, skin absorption of chlorhexidine from either chlorhexidine base-carrier suspension or chlorhexidine digluconate solution was compared with the effectiveness of the skin antisepsis treatment.

Section snippets

Materials

Chlorhexidine base and PCL (mw: 42 500) was purchased from Sigma–Aldrich (St. Quentin, Fallavier, France). Hibitane® 5% is a commercial chlorhexidine digluconate solution and was provided by Zeneca (Cergy-Pontoise, France). Acetone, acetonitrile, sodium acetate and acetic acid were provided by Prolabo (Lyon, France). Phospholipids (Epikuron® 200) were obtained from Lucas Meyer (St. Maur des Fossés, France). Epikuron® 200 was a purified wax-like phosphatidylcholine of soybean origin, which

Formulation and characterization of chlorhexidine nanocapsules

Table 1 shows the determination of chlorhexidine base loading in PCL nanocapsules from interfacial polymer deposition following solvent displacement method in formulations A and B. Encapsulation efficiency of chlorhexidine base in PCL nanocapsules was 60% whatever the initial chlorhexidine base amount dissolved in the organic mixture. No free chlorhexidine base was detected in both formulations A and B likely probably due to its low water solubility.

PCS was used as a sizing technique to assess

Steric and electrostatic mechanisms of bacterial adhesion

Chlorhexidine base and its salts are widely used as antiseptic, disinfectant, pharmaceutical and cosmetic preservative and antiplaque agents. For skin antisepsis, chlorhexidine has been formulated as a 0.5% (w/v) solution in 70% (v/v) ethanol [21]. However, although chlorhexidine salts are bacteriostatic and bactericidal in low and higher concentrations to many Gram-positive and Gram-negative bacteria, residual antiseptic action on the skin after rinsing and drying was not proven. In our study,

Conclusion

Chlorhexidine base loaded PCL nanocapsules, prepared by interfacial polymer deposition following solvent displacement yields quite uniform size and stable positively charged nanocapsules with good encapsulation efficiency. This in vitro study showed that the encapsulation of chlorhexidine base into PCL nanocapsules maintained an antimicrobial activity against several bacteria suggesting a sustained release of chlorhexidine base from PCL nanocapsules into surrounding medium. Furthermore, a

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