Material method discussed here is different from research proposal as material and method proposed in it was taken from one paper which explains about the encapsulation of ciprofloxacin in PLA microsphere so by giving the reference of this paper material and method for the preparation of microspheres for chlorhexidine was proposed.
In this completely different method and materials have been mentioned because the paper from which it is taken explains about the preparation of nanocapsules of chlorhexidine which is used to penetrate SC and its action against Staphylococcus epidermidis, Staphylococcus aureus , Enterococcusfaecalis etc, hence this has been referred.
Eucalyptus oil can be used to cover the nanocapsule with the help of spray drying and might help in better penetration of nanocapsule.
Tan. H.L. et.al (2005)
Materials:
Chlorhexidine base , poly(ε-caprolactone) [PCL] (mw: 42,500) ,5% chlorhexidine digluconate solution ,Acetone, Acetonitrile, Sodium acetate, Acetic acid, Phospholipids, Purified wax phosphatidylcholine of soybean origin-[ it is consisted of phosphatidylcholine (min. 92%) and small amount of phospholipids (lyso-phosphatidylcholine max. 3% and other phospholipids max. 2%]. Total content of fatty acid should be around 63%, non-ionic surfactant (polyoxyethylene 20 sorbitan monooleate, Tween 80), caprylic/capric triglyceride PEG-4 esters, Sodium salt of Carboxymethylcellulose (mw: 550000).
Mueller-Hinton agar plates and nutrient broth (pancreatic peptone of casein 0.1% in saline solution), Staphylococcus epidermidis, Staphylococcus aureus , Enterococcusfaecalis, Enterococcushirae , Enterobacteraerogenes, Pseudomonas aeruginosa, Pseudomonas aeruginosa , Escherichia coli . Porcine ear skin, adhesive tape strips (1.9 cm×5.0 cm).
Methods:
Preparation of chlorhexidinenanocapsules:
Chlorhexidine base loaded and unloaded PCL nanocapsules can be prepared by the interfacial polymer deposition following solvent displacement method. Dissolve 125 mg of PCL and 50 mg of Phospholipids (wax-like phosphatidylcholine) in 25 ml of acetone along with either 100 mg of chlorhexidine base (formulation A) or 25 mg of chlorhexidine base (formulation B) and this then should be dissolved in 0.5 ml of caprylic/capric triglyceride PEG-4 ester, at 40°C. Then, this mixture should be poured into 50ml of 0.15%(m/v) aq. surfactant( Tween 80), with light magnetic stirring. The resulting mixed phase is expected to turn milky with a bluish opalescence (i.e. Tyndall effect) as a result of the instantaneous formation of nanocapsules. Formulation C is prepared without chlorhexidine base supplementation (unloaded PCL nanocapsules).
Encapsulation efficiency:
To determine chlorhexidine base loading in PCL nanocapsules, a 10-ml PCL nanocapsule suspension should be first filtered through a sintered filter (porosity: 4 mesh; size: 9-15 μm) which retains the unencapsulated, precipitated chlorhexidine base. Then, 400 μl of the filtrate should be filtered through an ultrafree MC unit ( 10000 mw) and this then subjected to centrifugation for 5min. Finally, the chlorhexidine base amount (Q) in the 10-ml PCL nanocapsule suspension, the unencapsulated precipitated chlorhexidine base and the free chlorhexidine base in the ultrafiltrate can be assayed using high-performance liquid chromatography . Chlorhexidine assays were carried out by reversed-phase adsorption chromatography.Mixture of acetonitrile and 30 mM sodium acetate buffer (1:1, v/v) adjusted to pH 3.3 with acetic acid (96%) can be used as a mobile phase. Up to 40 μl of volume should be injected and at 260 nm detection can be performed.
The encapsulation efficiency of chlorhexidine base in PCL nanocapsules can be calculated through mass balance as follows
Particle size measurement and surface charge
Particle profiles like z-average diameter and polydispersity index (PI), as well as the zeta potential of nanocapsules can be determined by using photon correlation spectroscopy (wavelength=670 nm) and electrophoretic measurements
In vitro antimicrobial activity:
Minimum inhibitory concentrations (MICs) of 0.15% (m/v) chlorhexidine base loaded PCL nanocapsule suspension (i.e. 15 mg of chlorhexidine base encapsulated in 10 ml of nanocapsule suspension), unloaded PCL nanocapsule suspension (Formulation C) and 0.15% (m/v) chlorhexidine digluconate solution ( 5% solution in sterile water) compared for Staphylococcus epidermidis, Staphylococcus aureus , Enterococcusfaecalis, Enterococcushirae , Enterobacteraerogenes, Pseudomonas aeruginosa, Pseudomonas aeruginosa , Escherichia coli can be determined as follows:
Mueller-Hinton agar plates containing a range of concentrations (150-0.29 mg/l) of either chlorhexidine base loaded or unloaded PCL nanocapsules or chlorhexidine digluconate aqueous solution in sterile water inoculated with 1 μl of nutrient broth culture of test strains containing 1×108 CFU/ml. Incubated plates at 37°C for 24 h, and the surviving bacteria then to be counted after 24 h. MICs are then to be calculated. MICs of 0.15% chlorhexidine base loaded PCL nanocapsules stored in suspension at 4°C for 40 days were also determined.
Ex vivo anti microbial activity:
The recently developed ex vivo test was adapted for testing the antimicrobial activity of chlorhexidine base loaded PCL nanocapsules against Staphylococcus epidermidis on porcine ear skin. Porcine and human skin have similar surface lipids, barrier thickness and morphological aspects and thus excised porcine skin was expected to be useful for estimation of ex vivo human skin penetration behaviour
Freshly excised porcine ear skin samples from sacrificed animals should be cut carefully with a scalpel. Skin samples (2.54 cm2) are then to be mounted onto vertical diffusion cells containing saline solution in the receptor compartment. Then, 200 μl of either formulations A or C suspended in CMC hydrogel (0.4%, w/w), or 1% chlorhexidine digluconate solution to be applied with a sterile tuberculin syringe onto the delimited area skin in the donor compartment for 3 min. Close the top of sample compartment with a cap. To evaluate the effectiveness of residual chlorhexidine skin concentration against Staphylococcus epidermidis, 200 μl inocula prepared from clinically isolated microorganisms should then be applied to the stratum corneum (SC) surface for 4 h. Then, this inoculum is removed from the SC surface and 100 μl inocula are then be sown on a Mueller-Hinton agar surface. Again, 200 μl of fresh inocula should be re-applied to the SC surface for 4 h . Then, again the inoculum should be removed from the SC surface and sown on a Mueller-Hinton agar surface . Finally, 200 μl of fresh inocula again re-applied to the SC surface for 4 h , then the inoculum to be removed, and the SC surface then plated onto Mueller-Hinton agar contact plates for 1 min. Mueller-Hinton agar plates then to be incubated at 37°C for 48 h and the colonies observed should be visually counted. The survival rate of bacteria were expressed as CFU.
Chlorhexidine absorption into the SC:
To determine the diffusion of chlorhexidine through the SC, the outermost layer of epidermis, the SC should be removed by using the adhesive tapes to strip it from porcine ear skin treated for 3 min with either 0.60% chlorhexidine base loaded PCL nanocapsules suspended in hydrogel or 1% chlorhexidine digluconate solution. The adhesive tapes are then to be immersed for 30 min in 10 ml of acetonitrile-30 mM sodium acetate buffer (1:1, v/v) adjusted to pH 3.3 with acetic acid (96%). Assay of these solutions should be carried out for their chlorhexidine content by HPLC.
Lboutounne. H. et. al (2002)
REFERENCES:
Lboutounne.H., Chaulet.J., Ploton.C, Falson.F., Fabrice Pirot (2002) Sustained ex vivo skin antiseptic activity of chlorhexidine in poly(ε-caprolactone) nanocapsule encapsulated form and as a digluconate. Journal of Controlled Release, 82, 319-334
Tan, L. H. , Chan, L. W. andHeng, P. W. S.(2005) 'Effect of oil loading on microspheres produced by spray drying', Journal of Microencapsulation,22, 253 — 259
