This study demonstrates that 2% (wt/vol) chlorhexidine, the antiseptic agent recommended within EPIC and CDC guidelines for skin antisepsis prior to central venous catheter insertion, poorly permeates into deeper layers of the skin after 2 min and 30 min of exposure to the antiseptic. The concentrations of CHG within the upper 100-μm sections of skin were 0.157 (± 0.047) μg/mg tissue and 0.077 (± 0.015) μg/mg tissue after 2 min and 30 min, respectively. If 1 g of tissue is estimated to equal 1 ml, these levels are higher than the concentrations required to kill many common skin microorganisms, such as Staphylococcus epidermidis
, under in vitro conditions (10
). Below 300 μm, the CHG concentration remained less than 0.002 μg/mg tissue, which may not be effective for eradicating microorganisms on the skin (17
), especially microorganisms residing deep in the hair follicles. Furthermore, chlorhexidine activity is reduced in the presence of organic compounds, such as fatty acids, and at lower pHs (16
) and therefore may reduce the efficacy of skin antisepsis with CHG. An exposure time of 2 min was used to reflect the clinical conditions used prior to surgery (5
). Although the 2-min study appears to show a larger amount of bound chlorhexidine than the 30-min study, there is variability in concentrations measured in the top layers, as is expected with the shorter exposure period (24
), and the difference between 2 min and 30 min of exposure is not significant (P
> 0.05). It is likely that a steady state has not yet been reached at 2 min. A similar phenomenon was reported by Wagner et al. (23
). Skin was also exposed to 2% (wt/vol) CHG for 24 h, and the concentration of CHG in the deeper sections, i.e., beyond 300 μm, was less than 1 μg/mg tissue. These levels of CHG are more than the minimum bactericidal concentrations for many skin commensals (10
); however, this level of CHG was obtained only after a prolonged time of contact of the skin with CHG. In this study, no detectable levels of CHG were recovered from the receptor compartment, suggesting that aqueous CHG does not permeate through the full thickness of excised skin and is retained within the tissue. These results support previous research on another CHG-based compound, chlorhexidine phosphanilate, which was also shown not to permeate through full-thickness skin samples (25
In this study, a model for studying the delivery of CHG into excised full-thickness human skin was evaluated. Skin permeation studies are commonly performed in vitro with vertical or horizontal diffusion cells using skin or artificial membranes. This study was performed using vertical diffusion cells (Franz-type diffusion cells) to evaluate the delivery of CHG through excised full-thickness human skin. Such conditions mimic the in vivo environment by maintaining the physiological receptor fluid at body temperature and the skin surface temperature at 32°C (6
). Skin permeation studies generally evaluate drug delivery through the skin by measuring drug diffusion into the receptor fluid through the SC or epidermis, which are the main barriers for skin permeation. However, the use of stripped skin layers, such as isolated SC or epidermal layers, for drug permeation studies may influence the results, with possible retention of the drug in the dermal layers of the skin. Full-thickness skin was used in this study to determine the location of CHG throughout the skin, rather than studying the flux of the drug through the barrier layers. Following exposure to CHG, the full-thickness human skin was sectioned to a depth of 1,500 μm by sequential sectioning with a microtome, producing a total of 60 sections per skin sample. Skin sectioning has been used in many previous studies (21
); however, the SC is often removed by tape stripping prior to sectioning of the skin. In this study, the full-thickness skin samples were sectioned throughout the sample without prior removal of the surface layers. This study demonstrates that the CHG permeation through the full-thickness skin was not linear, which was expected due to the variation in structure at various layers. The top 100-μm layer of the skin, which contains SC (average of 10 to 20 μm thick) and other epidermal layers (50 to 100 μm thick), contained the largest amount of CHG following exposure to 2% (wt/vol) CHG over all time points studied. Previous research has shown that the main permeation barrier for skin absorption is the SC (3
), which is thought to be due to its high-lipid matrix and packed layers of keratinized epithelial cells. Furthermore, this study found that below 300 μm, at the dermal layer, the level of CHG remained constantly low. Depending on the body site, dermis contains hair follicles and other skin appendages, including sebaceous glands and sudoriferous glands (sweat-producing glands), which are of interest in skin antisepsis since they may be niches for microbial colonization of the skin following skin antisepsis (7
). It is generally recognized that skin antisepsis does not sterilize the skin; our study confirms this and demonstrates that it may be due to poor permeation of chlorhexidine into the deeper layers of the skin.
In conclusion, this study showed poor permeation of chlorhexidine through excised full-thickness human skin after 2 min and 30 min of exposure to aqueous 2% (wt/vol) CHG. The levels of CHG were highest within the top 100-μm sections of skin and remained consistently low within the deeper layers. Furthermore, the model presented in this study is a valuable tool in determining a permeation profile for chlorhexidine through human skin in vitro. This study lays the foundation for further research within this area with a view to potentially adopting alternative strategies for enhanced skin antisepsis in clinical practice.