Polyhexanide-Releasing Membranes for Antimicrobial Wound Dressings: A Critical Review

Abstract

The prevalence of chronic, non-healing skin wounds in the general population, most notably diabetic foot ulcers, venous leg ulcers and pressure ulcers, is approximately 2% and is expected to increase, driven mostly by the aging population and the steady rise in obesity and diabetes. Non-healing wounds often become infected, increasing the risk of life-threatening complications, which poses a significant socioeconomic burden. Aiming at the improved management of infected wounds, a variety of wound dressings that incorporate antimicrobials (AMDs), namely polyhexanide (poly(hexamethylene biguanide); PHMB), have been introduced in the wound-care market. However, many wound-care professionals agree that none of these wound dressings show comprehensive or optimal antimicrobial activity. This manuscript summarizes and discusses studies on PHMB-releasing membranes (PRMs) for wound dressings, detailing their preparation, physical properties that are relevant to the context of AMDs, drug loading and release, antibacterial activity, biocompatibility, wound-healing capacity, and clinical trials conducted. Some of these PRMs were able to improve wound healing in in vivo models, with no associated cytotoxicity, but significant differences in study design make it difficult to compare overall efficacies. It is hoped that this review, which includes, whenever available, international standards for testing AMDs, will provide a framework for future studies.

Keywords: PHMB; antimicrobial; controlled drug release; cytotoxicity; membrane; poly(hexamethylene biguanide); polyhexamethylene biguanide; polyhexanide; wound dressing; wound healing.

Figure 1

Methods commonly employed in the preparation of membranes for wound dressings.

Figure 2

Synthesis of poly(hexamethylene biguanide) hydrochloride (PHMB.HCl) via polycondensation of 1,6-hexamethylenediamine and sodium dicyanamide. n = 2–42.

Figure 3

Terminal groups that may occur in poly(hexamethylene biguanide) (PHMB) chains. (a) Amino; (b) cyanoguanidino; (c) guanidino; and (d) cyanoamino.

Figure 4

Schematic representation of the methods employed in loading a drug into a membrane. (a) Drug loading by soaking, in which the membrane to be loaded is immersed for a certain period in a drug solution, usually under agitation. It is the most straightforward method, but the loading yield can be low, as drug entry into the membrane occurs by diffusion across a concentration gradient. As such, some of the drug present in the solution will not be loaded. Loading yields depend not only on drug concentration, but also on other loading conditions (such as solvent, temperature, pH, ionic strength and time), as well as on drug/membrane interactions, molecular size of the drug, surface area, porosity and pore size of the membrane. (b) Drug loading by impregnation, in which the drug solution is added to the surface of the membrane to be loaded, being fully absorbed. (c) Drug loading by addition, in which the drug is added to the formulation employed to prepare the membrane. The drug must be able to withstand the conditions employed in membrane preparation and, usually, it has to dissolve in the formulation. With this method, non-toxic materials and reagents have to be employed, since extensive drug loss would occur if the final drug-loaded membrane was washed to extract cytotoxic leachables.

Figure 4

Schematic representation of the methods employed in loading a drug into a membrane. (a) Drug loading by soaking, in which the membrane to be loaded is immersed for a certain period in a drug solution, usually under agitation. It is the most straightforward method, but the loading yield can be low, as drug entry into the membrane occurs by diffusion across a concentration gradient. As such, some of the drug present in the solution will not be loaded. Loading yields depend not only on drug concentration, but also on other loading conditions (such as solvent, temperature, pH, ionic strength and time), as well as on drug/membrane interactions, molecular size of the drug, surface area, porosity and pore size of the membrane. (b) Drug loading by impregnation, in which the drug solution is added to the surface of the membrane to be loaded, being fully absorbed. (c) Drug loading by addition, in which the drug is added to the formulation employed to prepare the membrane. The drug must be able to withstand the conditions employed in membrane preparation and, usually, it has to dissolve in the formulation. With this method, non-toxic materials and reagents have to be employed, since extensive drug loss would occur if the final drug-loaded membrane was washed to extract cytotoxic leachables.

Figure 5

Schematic representation of the permeability cells specified for the measurement of moisture vapor transmission rate (MVTR) by standards (a) ASTM E96-90 (Payne permeability cup) and (b) EN 13726:2 (Paddington permeability cup). Both cups are depicted in the upright (left) and inverted (right) orientations. In the ASTM E96-90 standard, water evaporation through a test membrane is measured gravimetrically, by employing a Payne permeability cup located in a chamber at 23 °C or 32.2 °C and at 50 ± 2% relative humidity (RH). The Payne permeability cup contains water and its mouth is sealed with the test membrane, leaving a 19 ± 6 mm gap between the water surface and membrane. When the Payne permeability cup is placed in an upright orientation, this assay is designated “Procedure B—Water Method” or “Procedure D—Water Method”, depending on whether it is carried out at 23 or 32.2 °C, respectively. When in the inverted orientation, it is designated “Procedure BW—Inverted Water Method” and is carried out at 23 °C only. This assay can also be similarly carried out, but anhydrous CaCl₂ is used as a desiccant inside the upright Payne permeability cup, instead of water, leaving a 6 mm gap between the sample and desiccant. In this case, the assays are designated Procedures A, C or E—Desiccant Method, depending on the temperature at which it is carried out (23 °C, 32.2 °C or 37.8 °C, respectively). In the EN 13726-2 standard, the assay is similar to the ASTM assay, with the main differences lying in the use of a larger permeability cell (Paddington permeability cup), a smaller air gap between the water surface and sample (5 mm), a different temperature (37 °C) and RH (<20%), the use of samples preconditioned at 20 ± 2 °C and 60 ± 15% RH and the absence of a method that uses a desiccant.

Figure 6

Schematic representation of a Franz diffusion cell, as employed in the study of drug release from a single face of drug-loaded membranes.

Figure 7

Schematic representation of in vitro dynamic biofilm models employed in the evaluation of the antibacterial activity of AMDs. (a) The flatbed perfusion biofilm model is composed of a cellulose matrix that contains a biofilm held on an inclined glass slide. Growth media are perfused through the cellulose matrix and collected for bacterial quantification. (b) The colony drip-flow biofilm model is composed of a semipermeable polycarbonate membrane in which biofilms are grown, which is placed on an absorbent pad sitting on an inclined glass slide. Growth medium is dripped on the microscope slide, perfusing the absorbent pad. (c) The Duckworth biofilm device is a flow device that contains wells with an agar disk fed with flowing growth medium from beneath, on top of which semipermeable cellulose-based disks can be placed and inoculated for biofilm growth. AMDs are assayed by direct application on the biofilm.

Figure 8

Schematic representation of the in vitro assays employed in the evaluation of the wound-healing capacity of PRM6. (a) Wound-scratch assay, in which a linear gap is created in a cell monolayer by scratching the monolayer with the tip of a sterile pipette tip. The WD is assayed by applying extracts in cell culture medium to the cell monolayer. (b) Boyden’s chamber assay (also known as transwell migration assay or chemotaxis assay), in which a cell suspension is contained in hanging inserts with a porous membrane that are immersed in cell culture medium. The WD is assayed by applying extracts in cell culture medium to wells. After incubation, cells that migrate into the well are counted.