Using the Kull equation to analyze the biofilm viability experimental results, the combination of PHMB, EDTA salts, and VD had been found to provide synergistic, not simply additive, antibiofilm effectiveness in this report. The Kull equation was created to determine synergy of antifungal mixtures that were tested against planktonic fungi. Subsequently, Schmaus et al32 used the Kull equation for synergy determination of mixtures of 1,2 alkane diols as antimicrobial agents when tested on planktonic microorganisms. To the authors’ knowledge, the use of the Kull equation to prove antibiofilm synergy for any application, including wound cleansers, has not previously been published.
Biofilm infections persist in wounds as a result of biofilm EPS blocking access of antimicrobial agents to their sites of action as well as microbes in biofilm having depressed metabolism and activated protective stress responses.33-36 Thus, the removal of biofilm from wounds is important to promote wound healing,37 and an aggressive multimodal therapy that includes debridement, frequent lavage, and antimicrobial treatment is supported by clinical evidence.35,37 Such repeated attacks on biofilm forces it to reattach and reform, temporarily driving it into an immature state more susceptible to host defenses and antimicrobials.12,38 However within hours posttreatment, biofilm can reform39 and spread into tissues where it adheres firmly,2 thus repeatedly applied debridement40 and topical biocides have been used to deter biofilm from reforming and entrenching into tissue.2,35 Of note, microbes do not proliferate unchecked in healthy tissue.41 This suggests that a multifaceted approach is required in vivo to remove all causes of tissue damage, such as compromised circulation, edema, or repeated trauma, while protecting injured tissue from microbial invasion and/or biofilm formation.42
As listed in Table 1 and, discussed further on, PHMB, EDTA, and the VD each individually are known to disrupt EPS and permeabilize microbial cell membranes and impair processes needed for viability.
Polyhexamethylene biguanide is a broad-spectrum antimicrobial, biocompatible43,44 cationic polymer that has a low average molecular weight range of 2000 to 4500 Daltons. Polyhexamethylene biguanide can penetrate EPS and form polyelectrolyte complexes with polyanions, such as DNA and polysaccharides, causing flocculation and large aggregates that may be removed more easily.45 When PHMB is present, aggregation of EPS polyanions (alginates) in P aeruginosa biofilm is visually observable as “clumps.” Bueno and Moraes46 used this effect to bind PHMB to chitosan-alginate wound dressings for sustained release of PHMB. Once in contact with microbes, the polycation PHMB interacts with microbial membrane anions to disrupt the cell membrane with resultant leakage of cytoplasmic components and inhibition of membrane-bound enzymes17,47,48; additionally, PHMB enters bacterial cells, condenses chromosomal DNA, and arrests cell division.49 Also, PHMB has been recommended as an antimicrobial compound of choice for chronic wounds and burns.50
Ethylenediamine tetraacetic acid sodium salts (di- and tri-) functions over a wide pH range (2–12), which is a requirement as both the wound milieu pH range can be from acidic to basic depending on native biochemical processes for wound healing51 and multispecies biofilms (aerobic, facultative, and anaerobic) can have pH gradient ranges from acidic to basic.52 The EDTA is known to chelate divalent metal cations essential for bacterial growth53 and destabilize bacterial membranes and matrix integrity.18,53-55 As an example, EDTA is reported to potentiate the antimicrobial effects of quaternary ammonium compounds by extraction of lipopolysaccharide from P aeruginosa cell walls.56 Cationic quaternary ammonium compounds absorb on negatively charged cell walls concurrently with EDTA chelation of cell wall metal cations with a resulting loss of lipopolysaccharides and increased cell permeability.56 Also, EDTA is reported to inhibit excess matrix metalloproteases by chelating zinc and calcium,57 thereby facilitate wound healing.58 Consistent with healthy human tissue, the wound cleanser’s EDTA (di- and tri-sodium) provides a slightly acidic pH, which, together with physiologic osmolarity to prevent cell dehydration from high osmolarity or cell swelling from low osmolarity, contribute to mitigating pain while being non-cytotoxic to human tissue.59,60
The VD, ethylhexylglycerin and octane-1,2-diol, are multifunctional personal care ingredients commonly used in underarm deodorants, with moisturizing and antimicrobial activities resulting in odor reduction due to inhibition of odor-causing Gram-positive bacteria (eg, Corynebacterium spp, Leifsonia aquaticum, Ochrobactrum anthropi, Kocuria rhizophila).61-64 They are known to disrupt microbial membranes and synergistically boost the effectiveness of preservatives such as parabens or phenoxyethanol19 — eg, ethylhexylglycerin potentiated the lethality of phenoxyethanol against P aeruginosa and Aspergillus niger.65 With 8 carbon atoms and 2 hydroxyl groups on adjacent carbons, these amphiphilic surfactants, with an hydrophilic-lipophilic balance (HLB) of 7 to 7.5, provide humectance (hydration) and emollience (occlusivity, softening, lubrication, spreading, and delivery of actives), solvate out lipids and humic components (ie, from biofilm), and have antimicrobial activities differing from PHMB.63,64 While PHMB has broad-spectrum activity against bacteria, fungi, protozoa, and viruses, the VD are particularly effective against Gram-positive bacteria and yeasts.65,66
By incorporating the combination of PHMB, EDTA, and VD, synergistic antibiofilm effectiveness was found, not just additive effectiveness. The SI values determined were 0.148 with P aeruginosa and 0.032 with MRSA. The SI values are notably lower than 1, thus indicating high synergy.
The base solution for these studies and used in the wound cleanser was developed to complement the synergistic antibiofilm efficacy of the 3 components (PHMB, EDTA, and VD). The base solution comprises water, a salt, a mucoadhesive, and a surfactant.
Surfactants lower the interfacial tension between substances and can loosen and remove dirt, debris, slough and loosen biofilm from the wound. Very hydrophilic surfactants, such as poloxamer 188 with an HLB of 29, have been used to aid in the removal of biofilm from wound surfaces.12,67,68 However, the nonionic surfactant, P-407 in the base solution, not only lowers interfacial tension to aid in removal of debris but also is a detergent (HLB 18–21.5)69 that incorporates (ie, emulsifies) hydrophobic materials into water.70 These hydrophobic materials may be present as lipids, proteins, and polysaccharides, for instance. By incorporating these organic substances into water, they are easier to remove by irrigation. In addition to serving as a detergent and surfactant, P-407 aids in the solubility of the VD due to its amphiphilic competency.71 Poloxamer 407 (Pluronic 127; Sigma-Aldrich) is a triblock copolymer consisting of a central hydrophobic block of about 101 repeats of polypropylene glycol flanked by 2 hydrophilic blocks of about 56 repeats of polyethylene glycol. Of note, P-407 helps to maintain the activity of PHMB17 and VD.
Mucoadhesives are used to increase residence time of a composition on a mucosal membrane such as found in the gastrointestinal tract, lungs, and eyes. Mucosal membranes contain up to 95% water, with the remaining components comprising glycoproteins, lipids, and other hydrophilic organic matter.72 In general, a wound bed has similarities to mucosal tissue, such as higher water content combined with the presence of hydrophilic organic matter, as found in wound exudate as well as biofilm. Therefore, the mucoadhesive, water-soluble, neutral-charged HPMC73 forms a hydrated film on the wound surface and, hence, increases the cleanser’s residence time.
In order to adjust osmolality to a physiologically normal range (290–320 mOsm/kg), sodium chloride is added as a component of the base solution. When products used on wounds are hypertonic, water is pulled out of surrounding tissue through osmosis, which causes dehydration and cell size shrinkage.74 The opposite effect occurs when osmolality is hypotonic; the surrounding tissue pulls in water causing cell size enlargement.74 Pain is created with either too high or too low osmolality, and neurological damage is suffered.74 Therefore, a physiologically balanced osmolality is preferred for protection of healthy tissue in and surrounding the wound.
In summary, the targeted points of the wound cleanser are (1) synergistic antibiofilm components (PHMB, EDTA, and VD) complemented by (2) P-407 to remove loose debris from the wound surface through interfacial tension reduction as well as to incorporate hydrophobic materials (ie, emulsification) into water and (3) the mucoadhesive HPMC to increase residence time on wound surfaces. Additionally, physiologically balanced pH and osmolality are gentle to human tissue.59,60,74
Clinical validation of the antimicrobial cleanser is in progress with bacterial fluorescence and DNA sequencing. This also includes targeted data points of wound healing progression and economic evaluation. The algorithm for use of this wound cleanser is provided under the guidelines from the International Wound Infection Institute International Consensus Update 2016/Wound Infection in Clinical Practice for “Effective Wound Infection Management,”75 which recommends regular wound evaluation for signs of infection and to “cleanse the wound with each dressing change.” The cleanser effectiveness may be impacted by enzymes, ointments, or oils in the wound bed. Irrigation of the wound bed may be performed with the cleanser to thoroughly rinse the wound bed from these agents.