The development of technology for preparing chitosan–protein scaffolds loaded with lysostaphin, which potentially could be used as dressing for wound treatment and soft tissue infections caused by Staphylococcus aureus.
The development of technology for preparing chitosan–protein scaffolds loaded with lysostaphin, which potentially could be used as dressing for wound treatment and soft tissue infections caused by Staphylococcus aureus.
The unique technology of chitosan solubilization using gaseous CO2 instead of organic or inorganic acids was used for the incorporation of lysostaphin, the enzyme that exhibits bactericidal activity against staphylococci, within the structure of chitosan–protein sponges. The developed chitosan–protein scaffolds loaded with lysostaphin revealed high antistaphylococcal activity, which has been confirmed with a large (n = 143) collection of clinical (skin and wound infections) and animal (bovine mastitis) isolates of these bacteria, including MRSA. No change of bactericidal activity of the lyophilized materials has been observed during half-year storage at 4°C.
The developed materials are potential candidates for preparing biologically active, antistaphylococcal wound dressing materials.
Staphylococci belong to the most popular and most burdensome aetiological factors of wound and soft tissues infections. The developed chitosan-protein scaffolds loaded with lysostaphin could be a possible solution to problems associated with treatment of these infections.
Fast and effective treatment of wounds constitutes one of the most important challenges of modern medicine. In the United States, chronic wounds alone affect around 6·5 million patients per year, with annual treatment costs rising up to 25 billion USD (Sen et al. 2009). The most important factors affecting the wound healing, significantly delaying this process, are bacterial and fungal infections, and the most common micro-organisms isolated from both acute and chronic wounds of various aetiologies are as follows: Staphylococcus aureus, Pseudomonas aeruginosa, β-haemolytic streptococci and also Candida albicans. Infections caused by Staph. aureus are especially difficult to treat. This group of bacteria has evolved resistance to a plethora of antibiotics currently in use for human therapies and has also developed biofilm-forming ability, which significantly reduces antibacterial activity of antibiotics and disinfectants. The most worrisome problem is the rapidly growing number of methicillin-resistant staphylococci, which are designated as MRSA (Methicillin-Resistant Staph. aureus). The MRSA isolates, including strains isolated from wounds, often exhibit multi-drug resistance (MDR) and are simultaneously not susceptible to many antibiotics from different chemical groups with different mechanisms of activity (Fry 2013; Goyal et al. 2013). Therefore, there is a great need for novel, nonantibiotic chemotherapeutics with marked antistaphylococcal activity. In the case of wound healing, it is also very important to develop an effective delivery method for antimicrobial agents at the infection site.
The aim of the present study was to develop chitosan–protein scaffolds loaded with lysostaphin, which could be used as bactericidal, antistaphylococcal, wound dressing materials. Many previous reports revealed that using natural biopolymers as components of dressings provides favourable conditions for cell proliferation, tissue regeneration and finally healing of the wound (Ma 2008; Cui et al. 2010; Gorczyca et al. 2013). Our choice of lysostaphin as an antistaphylococcal agent was not accidental. Lysostaphin is an enzyme with bactericidal activity against Staph. aureus and other staphylococcal species (Schindler and Schuhardt 1964). The target of the lysostaphin activity is the pentaglycine interpeptide bridges of the unique staphylococcal peptidoglycan (Schleifer and Kandler 1972; Iversen and Grov 1973). Other Gram-positive and Gram-negative bacteria, including the normal microbiota of human and animals' skin, are not susceptible to this enzyme (Schindler and Schuhardt 1964). The unique biological activity of lysostaphin presents numerous possibilities for applications of this enzyme as an antistaphylococcal agent in human and animal therapies. The high therapeutic potential of the enzyme has been confirmed in many animal models of staphylococcal infections, including endocarditis (Climo et al. 1998) and ocular infections (Dajcs et al. 2000). Several reports have also shown that lysostaphin is a promising agent in the eradication of staphylococci biofilms from biotic and abiotic surfaces including medical devices such as catheters (Wu et al. 2003; Walencka et al. 2005, 2006). Recently, lysostaphin has been successfully tested as a bactericidal, antistaphylococcal agent in wound dressing materials (Cui et al. 2010; Miao et al. 2011) and in meshes used in herniorrhaphy (Belyansky et al. 2011).
Herein, we have described a novel technology for the preparation of chitosan–protein scaffolds loaded with lysostaphin. The in vitro analysis of antistaphylococcal activity of the produced materials confirmed their high potential for use as wound dressing and treatment of soft tissue infections caused by Staph. aureus. Moreover, in our opinion, the prepared biopolymer matrix has potential application as a universal vehicle to deliver any other protein or peptide with antimicrobial activity to the infected tissue location.
Chitosan and all buffers were purchased from Sigma (Seelze, Germany). Genipin was bought from Challenge Bioproducts (Yun-Lin Hsien, Taiwan), and media for growing bacteria, LB broth, LA agar and Baird-Parker agar, were supplied by BioMaxima (Gdansk, Poland). Fish proteins used in this work were extracted from salmon skin (Salmo salar). The extraction was performed according to the procedures described earlier by Kolodziejska et al. (2008).
The recombinant lysostaphin was produced in the cells of Escherichia coli TOP10F' strain (Invitrogen, Carlsbad, CA), transformed with the plasmid pBAD2Lys and constructed earlier in our laboratory (Szweda et al. 2007). The production of the enzyme was carried out in a 5-l bioreactor (Biostat C; Braun Co., Germany) according to the procedure optimized by Szweda et al. (2014a). Enzyme purification was conducted with the use of metal affinity chromatography on a Ni-NTA His-Bind Resign column (Novagen, Madison, WI).
Chitosan–protein sponges were formulated according to a previously described method (Gorczyca et al. 2013). Briefly, 1·5 g of chitosan powder was suspended in 100 ml of 0·5 mol l−1 acetic acid. The dissolved chitosan was precipitated from the suspension by dropwise addition of 0·5 mol l−1 sodium hydroxide, followed by centrifugation at 4000 g for 30 min. The resulting precipitate was washed twice with distilled water, centrifuged again at 4000 g for 30 min, suspended in distilled water in an amount necessary to obtain a total weight of 80 g, homogenized and saturated with CO2 for 3 h at room temperature with the use of a hollow shaft stirrer to obtain a clear aqueous chitosan solution. The genipin ethanolic solution and lysostaphin in phosphate buffer (pH 7·4) were next added to final concentrations, as reported in Table 1. The mixture was stirred for 10 min and cooled to <12°C. Subsequently, the collagen–gelatin solution, prepared in parallel by dissolution of 1·2 g of gelatin in 20 ml of distilled water and homogenized after the addition of 0·6 g collagen, was added to the chitosan–genipin–lysostaphin blend, and vigorous stirring was continued for 10 min. The resulting blend was poured into moulds (12 × 12 × 0·4 cm), incubated for 24 h at 12°C, frozen at −20°C, freeze-dried, conditioned at room temperature and <90% humidity for 3 h and stored in closed plastic bags at 4°C. The gelatin was used as a component of the produced scaffolds to minimize the future production costs of the final material (much cheaper than collagen). Complete replacement of collagen with gelatin is not possible, due to much higher positive influence of collagen on wound healing process in comparison with gelatin.
|Genipin (%, w/w)||Lysostaphin (U ml−1)|
The disc diffusion and spectrophotometric assays were used for determination of bacteriolytic activity of lysostaphin and chitosan–protein materials incorporated with the enzyme. In the case of disc diffusion method, the discs (diameter = 10 mm and height = 3 mm) containing lysostaphin were placed on the Baird-Parker agar plates inoculated with the indicator strain of Staph. aureus. The plates were incubated at 37°C for 24 h. Following the incubation, the diameters of the growth inhibition zones were measured in millimetres. The basic spectrophotometric assay used for analysis of the enzyme solutions (generated during the analysis of release of the enzyme from the scaffolds) was performed according to the procedure proposed by Marova and Kovar (1993) with slight modifications. The reaction mixture, containing a 6-ml sample of an overnight culture of Staph. aureus ATCC 29213 cultivated in LA medium, diluted in 0·1 mol l−1 phosphate buffer (pH 7·4) to OD600 = 0·25, was pre-incubated at 37°C for 10 min, and then, different volumes of lysostaphin solutions were added. After 10 min of incubation at 37°C, the changes in turbidity of the reaction mixture were determined. One unit (U) of the lysostaphin activity was defined as an amount of preparation causing a 50% reduction in turbidity of the 6-ml cell suspension within 10 min at 37°C.
The slightly modified procedure was used for the direct determination of the activity of chitosan–protein scaffolds (containing the enzyme at a concentration of 10 U ml−1 and genipin in concentration of 0·5%). In this case, a disc (diameter = 4 mm and height = 3 mm) of the investigated material was placed in 3 ml of Staph. aureus suspension (OD595 = 0·5), and the decrease in the OD value of the cells suspension was tested at 5-min intervals. The test was carried out using a Victor 3 (Perkin Elmer, Waltham, MA) plate reader, in 12-well plates, at 37°C with constant shaking. Time after which the OD value dropped to zero was used as a parameter for comparison of activity of the material against the Staph. aureus strains tested (143 isolates).
The lysostaphin-impregnated scaffold discs were placed on the 1·5% agarose gel and incubated at 37°C for 18 h. Diffusion of the enzyme from the scaffold to the agarose gel was detected by the Commassie blue staining; the scaffold without the enzyme was used as a control.
For more advanced quantitative analysis of the lysostaphin release, the enzyme-impregnated scaffolds discs (diameter = 10 mm and height = 3 mm) were placed in 1·5 ml of phosphate buffered saline (pH 7·4) and incubated in a shaking incubator at 37°C. Samples (100 μl) were taken from the mixture every 10 min and replaced with fresh PBS (pH 7·4). The concentration of lysostaphin in the sample was analysed using the spectrophotometric assay described above.
A total of 143 Staph. aureus strains were used for the determination of bactericidal, antistaphylococcal activity of chitosan–protein scaffolds containing lysostaphin. Ninety-three strains were isolated from the milk of mastitic cows, from the collection of Department of Pharmaceutical Technology and Biochemistry of Gdansk University of Technology. Twenty strains were isolated from human skin and wound infections and were supplied by the Department of Clinical Bacteriology at the Provincial Hospital, Koszalin (Poland). Thirty strains were obtained from Department of Clinical Microbiology, Central Hospital, Vaxjo (Sweden) (Sjolund and Kahlmeter 2008). Six of the strains were classified as MRSA (all of them isolated from human skin and soft tissue infections).
Application of the original method allowed us to prepare materials in which most of lysostaphin was not chemically bound to biopolymer, chitosan–protein (collagen and gelatin) matrix. Consequently, the enzyme was easily released from the material to the wound site, which is crucial for eradication of staphylococci causing infection. Efficient release of lysostaphin from the material has been confirmed with disc diffusion and spectrophotometric assays. Scaffold discs (with 10 U ml−1 lysostaphin and without lysostaphin) were placed on the surface of 2·0% agarose gels, and diffusion of the lysostaphin from the material was detected by Commassie blue staining, as a blue zone appearing around the scaffold (Fig. 1a). In the case of the control material (without the enzyme), a clear blue zone was not observed (some colour identified in the location of the samples was the result of collagen and gelatin content in the material). (Fig. 1b).
Disc diffusion assay was also employed for confirmation of bactericidal, antistaphylococcal activity of the protein released from the materials. For this determination, the disc of scaffolds was placed on the surfaces of Baird-Parker agar inoculated with the reference strain of Staph. aureus ATCC 29213. The results of analysis carried out with materials of different composition, 1. constant concentration of genipin (0·5%) and increasing concentrations of lysostaphin (0, 10, 20 30 U ml−1); 2. constant concentration of the enzyme (10 U ml−1) and increasing concentrations of genipin (0·0, 0·5, 1·0, 2·0%), revealed an evident correlation between the efficiency of enzyme release—bactericidal activity of the scaffolds and concentrations of lysostaphin, and also genipin used for cross-linking of the biopolymers. The diameter of the growth inhibition zone of the reference strain of bacteria evidentially increased with the increase in the enzyme concentration (when genipin concentration was constant—0·5%) (Fig. 2a) and decrease of genipin concentration (when concentration of the enzyme was constant—10 U ml−1) (Fig. 2b).
In another experiment, scaffolds were washed with PBS, and the bacteriolytic activity of the washes was assayed spectrophotometrically. This approach allowed for a more detailed analysis of lysostaphin release from the scaffolds, especially determination of kinetics of this process. The results obtained generally confirmed previous observations that the release rate is dependent on the concentration of the cross-linking agent used for the scaffold preparation (Fig. 3) and on the enzyme concentration (data not shown). In the case of the scaffold produced without cross-linking (0% genipin), nearly 90% of the enzyme was washed out within 90 min. Using genipin in concentrations of 0·5, 1·0 and 2·0% resulted in a decrease of the amount of released enzyme, to the level of approx. 50, 40 and 30%, respectively, which in our opinion is still satisfactory. The analysis of the curves presented on Fig. 3 indicates that independent of the cross-linking agent concentration, the unbound enzyme, was washed out from the scaffold structure within about 30 min.
The lyophilized scaffold containing lysostaphin was stable under refrigerated conditions. Bactericidal activity of the sample stored at 4°C for 6 months was identical to that of the freshly prepared (detailed data not shown).
Resistance to lysostaphin is extremely rarely among Staph. aureus strains; however, the particular isolates differ in susceptibility to the lytic activity of this enzyme (Climo et al., Szweda et al. 2014b). Herein, we compared the bacteriolytic activity of the scaffolds containing lysostaphin against the population of 143 Staph. aureus strains isolated from human skin and wounds infections (n = 50, including 6 MRSA isolates) or from bovine mastitis (n = 93). As a parameter for comparison of activity of the fabricated scaffolds, we used the time period required for the reduction of turbidity of Staph. aureus cell suspensions from OD595 = 0·5 to OD595 = 0·0. As shown in Fig. 4, a slight difference was noted between strains of human and animal origin. Generally, strains isolated from bovine mastitis were more susceptible to the enzyme action. For most of the isolates (56 isolates, 60·2%), the cells were completely lysed within 30 min, and only four isolates (4·3%) required more than 65 min for the same effect. On the other hand, most of the strains of human origin required more than 30 min for complete lysis (usually 35–50 min); only six isolates (12·0%) were lysed within 30 min, and for 10 of them (20·0%), the time-to-lysis was longer than 60 min. The MRSA strains did not show any specific resistance to lysostaphin. One strain was completely lysed after 25 min, and two isolates required 45 min, while three strains underwent complete lysis within 55 min.
Wounds are easily infected with bacteria from various sources, including patients' and medical personnel's skin, medical equipment, but also from the air. The antibacterial protection afforded by conventional absorbent cellulose dressings has been shown to be limited, particularly in the presence of serous exudate that may compromise dressing integrity (Lavrence 1994). Thus, many other synthetic polymers and biopolymers have been extensively investigated as potential materials for management of wounds and burns. Among them, chitosan and especially collagen, selected for our research, are considered the most promising. The wound dressing materials produced on the basis of these biopolymers demonstrate numerous advantages: high biocompatibility, nontoxicity, capacity to absorb wound exudates, maintenance of the moisture, permeability to oxygen, partial protection against secondary infections (because the chitosan naturally characterizes with antimicrobial activity) and desirable mechanical properties (Jayakumar et al. 2011; Busilacchi et al. 2013). There are also many possibilities of chemical modifications of chitosan, which enables dressings to be prepared in the form of hydrogels, fibres, membranes, scaffolds and sponges (Jayakumar et al. 2011). The goal of our investigation was not only to prepare the new version of chitosan–collagen-based dressing, but the primary goal was to prepare a material that could be used as a vehicle to deliver an antistaphylococcal agent—lysostaphin—into the indolent wound.
To achieve this goal, we developed a unique fabrication technology of chitosan–protein scaffolds. In comparison with many other technologies described in the literature, the most important advantage of our proposition is complete elimination of inorganic or organic acids at the beginning of the process. In fact, in our technology, the acids are used only for initial solution of chitosan powder. Next, the solution of chitosan is neutralized with NaOH, and the precipitated microcrystalline form of chitosan is collected by centrifugation. Solution of microcrystalline chitosan for preparing scaffold is carried out with CO2, which is crucial for incorporation of lysostaphin into the structure of the biopolymer material while maintaining the biological activity of the enzyme. The standard technologies require using of organic (usually acetic acid) or inorganic acids for solution of microcrystalline chitosan. The acid is present in the reaction mixture throughout the whole process of the scaffolds fabrication, which is an obvious limitation from the point of view of incorporation of the biological active enzyme into the structure of the material. Even in the case of dressing material fabrication without the enzyme component according to standard procedures, the residual acid has to be removed from the final product. It is necessary because of the potentially harmful effects of organic acids on various cell populations as well as the impediment of the epithelialization process that is important in wound healing (Miller et al. 1992). In most cases, this is achieved by washing with water or alcohol solutions (Lin et al. 2009; Yan et al. 2010; Nandagiri et al. 2011). This step is followed by additional drying of the scaffolds, to obtain a dry product. Such procedures often lead to the loss of some of the incorporated enzyme or at least causes a partial decrease of its biological activity. Moreover, the loss of the original structure of the formulated materials as well as the partial dissolution of the scaffold is observed, rendering the procedure less reproducible. Another important advantage of the proposed technology is using genipin as a cross-linking agent instead of most popular glutaraldehyde. Genipin is a natural compound obtained from geniposide after enzymatic hydrolysis and is characterized as having lower toxicity in comparison with glutaraldehyde. The detailed systematic study of genipin cross-linking effects on chitosan/gelatin scaffolds has been recently described (Sarem et al. 2013).
The physicochemical advantages as well as good biocompatibility (low toxicity towards keratinocytes) of chitosan–protein materials produced according to the developed technology have been widely discussed in our previous publication (Gorczyca et al. 2013). The current investigation has confirmed that the material can be successfully applied as a vehicle to deliver antistaphylococcal proteins or peptides agents into the healing tissue. The particular agent used in this study was lysostaphin. However, we believe that other antimicrobial peptides or enzymes could be effectively delivered to the infected wound from the developed chitosan–protein scaffolds.
Especially, important seems to be the effective release of the active antibacterial protein from the biopolymer material and subsequent eradication of the staphylococci from the infected tissue. As it has been shown in the current study, the amount of the enzyme released from our novel material is affected by the concentration of the cross-linking agent, namely genipin used for its preparation. In the case of material produced without using the cross-linking agent, about 90% of the enzyme is released within 90 min. However, taking into account the requirements for the physicochemical properties of dressings, the materials with optimal parameters are produced when genipin is used at a concentration of 0·5%. In this case, about 50% of the lysostaphin is released when used at a concentration of 10 U ml−1, and the release rate is slightly lower with the decrease of lysostaphin concentration (data not shown). It clearly indicates that most of the protein is not chemically immobilized on the surface of biopolymer material, but rather is absorbed to the material. Lysostaphin has been widely tested as a potential therapeutic agent in many animal models; however, there have been only very few attempts of using this protein as a component of biologically active dressings for the treatment of human clinical skin infections. Miao et al. (2011) successfully used the enzyme for preparing cellulose dressing with antistaphylococcal activity. The authors modified the cellulose fibres to generate aldehyde groups for the covalent immobilization of lysostaphin. The resulting lysostaphin-functionalized cellulose fibres were further processed to obtain bandage preparations that showed activity against Staph. aureus in an in vitro skin model. The chitosan–collagen-based hydrogel dressing materials with immobilized lysostaphin was developed by Cui et al. (2010). The produced material exhibited advantageous physicochemical parameters, antistaphylococcal activity, and was successfully used in treatment of wounds in a New Zeeland rabbit model. However, the technology of its production was more complicated and time-consuming, in comparison with the technology proposed in the present study. Using lysostaphin for the treatment of wounds infected with staphylococci was also investigated by Desbois et al. (2010). The authors revealed that combination of lysostaphin with the antimicrobial peptide ranalexin exhibited synergistic antibacterial effect in the treatment of wounds infected with MRSA, in a rabbit model of wound infection. However, in that study, classical gauze was used as the wound dressing. The immobilization of lysostaphin was also applied for preparing meshes used in herniorrhaphy. Animals implanted with mesh containing lysostaphin had a dramatically improved rate of survival (Belyansky et al. 2011). It is worth mentioning that lysostaphin also exhibits moderate elastolytic activity. As elastin is an important component of the skin tissue. The ability of partial degradation of this protein could be important, from the point of view of better penetration of the enzyme into the skin tissue and an easier access to the bacterial habitat (Park et al. 1995).
Using a large collection of bacterial isolates, we have demonstrated the potential of lysostaphin for the treatment of staphylococci isolated from different sources, including skin infections. The obtained results revealed significant differences in susceptibility; however, none of the strains tested, including 6 MRSA isolates, could be considered resistant. The strains isolated from bovine mastitis used in this study were previously tested for susceptibility to 20 antibacterial antibiotics. The level of resistance was rather low; however, in the case of streptomycin, about 10% of isolates were not susceptible, and the resistance to β-lactam antibiotics was approx. 20% (Szweda et al. 2014b). It clearly indicates that lysostaphin, but also other enzymes with antimicrobial activity, especially peptidoglycan hydrolases, should be considered as an interesting alternative to antibiotics in the treatment of diseases caused by staphylococci, including skin or wound infections (Szweda et al. 2012). Development of several efficient technologies of production of lysostaphin additionally supports possibilities of application of this enzyme as antistaphylococcal agent (Mierau et al. 2005; Szweda et al. 2014a; Zhao et al. 2014).
The developed chitosan–protein scaffolds loaded with lysostaphin reveal high bactericidal, antistaphylococcal activity, which has been confirmed with a large (n = 143) collection of human clinical and animal isolates of these bacteria. Thus, these scaffolds are interesting candidates for preparing biologically active, antistaphylococcal wound dressings. Moreover, in our opinion, the developed chitosan–protein matrix could be used as a universal vehicle to deliver other peptides and proteins with antimicrobial activity into the indolent wounds.
This work was supported by grants no. from the Polish Ministry of Science and Higher Education N N405 420739 and N N312 258540 (MNiSzW) and the VENTURES programme established by the Foundation for Polish Science, co-funded by the European Regional Development Fund under the Operational Program Innovative Economy: Project No. VENTURES/2010-5/2.
The authors are grateful to Prof Randy Worobo from Cornell University for his help in preparing the English version of the manuscript of publication.
Authors do not declare any conflict of interest.