Bacillus subtilis in PVA Microparticles for Treating Open Wounds.

Open wound dressings should provide a moist environment, protect the wound from bacterial contamination, and shield it from further damage. These requirements, however, are hard to accomplish since such wounds are colonized by pathogenic bacteria, including resistant species such as methicillin-resistant Staphylococcus aureus (MRSA). A new approach for treating open wounds that is based on sticky and dissolvable polyvinyl alcohol (PVA) microparticles containing live Bacillus subtilis (B. subtilis) is described. Microparticles, fabricated by the spray-drying technique, were administered directly to an open wound while B. subtilis continuously produced and secreted antimicrobial molecules. B. subtilis in PVA microparticles demonstrated remarkable antibacterial activity against MRSA and S. aureus. In in vivo experiments, both B. subtilis and empty PVA microparticles were effective in decreasing healing time; however, B. subtilis microparticles were more effective during the first week. There was no evidence of skin irritation, infection, or other adverse effects during the 15 day postoperative observation period. This concept of combining live secreting bacteria within a supportive delivery system shows great promise as a therapeutic agent for open wounds and other infectious skin disorders.

Introduction

Skin healing is critical for maintaining the barrier between the body and the external environment. Hydration is considered to be the single most important factor for optimal wound healing by providing a protective barrier, reducing dermal necrosis, and accelerating wound re-epithelialization.[1] There is also an urgent need for effective infection control to promote rehabilitation and avoid systemic inflammation.[2] This requirement, however, is hard to accomplish since open wounds are invariably colonized by pathogenic bacteria including Staphylococcus aureus (S. aureus) and methicillin-resistant Staphylococcus aureus (MRSA), which express virulence factors.[3,4] Consequently, the preferred open wound dressing should not only provide a moist environment but also protect the wound from bacterial contamination, shield the wound from further damage, and promote granulation tissue formation.[5]

Currently available treatments for open wounds are often less than ideal as drug delivery systems. Antiseptics, for example, are toxic to human keratinocytes and fibroblasts and may increase the intensity and duration of skin inflammation.[2] The antibacterial activity of topical therapy lasts only up to 12 h, and dressing must be replaced routinely.[6] Topical formulations also suffer from inherent shortcomings, particularity those containing an oily phase such as ointment and emulsions. These eventually dry, and the dressing tends to stick to the surface of the wound, causing unnecessary pain, cell damage, and impaired wound healing.[7] Finally, oral and topical antibiotics have not always been shown to improve healing rates of chronic ulcers,[8] probably due to the rapid emergence of resistant bacteria.[9]

The objective of this study was to design and study a new class of topical formulae for treating open wounds. We hypothesized that a live bacterial formulation that continuously produces antibacterial molecules and delivers them directly to the open wound surface may be an alternative therapy. Our structure motif is based on incorporating Bacillus subtilis (B. subtilis), a Gram-positive bacterium that produces and secretes a variety of potent antibacterial agents,[10] into polyvinyl alcohol (PVA) microparticles. PVA was selected as the carrier due to its water solubility, hygroscopic properties, and excellent ability to adhere to the skin.[11,12] In addition, owing to its very low modulus of elasticity, PVA formulations cause minimal mechanical irritations, exhibit excellent biocompatibility with body tissues, and do not require removal or replacement.[13,14] From an industrial point of view, PVA is scalable and ideal for fabricating microparticles due to its semicrystalline structure and physical properties.[11,14]B. subtilis has been marketed as a biocontrol agent for various crop diseases thanks to its ability to protect against bacterial and fungal attack in plants.[15]B. subtilis has been designated Generally Regarded as Safe (GRAS) by the U.S. Food and Drug Administration.[16−19] In this study, we describe a particulate system that can be administered directly onto the open wound surface, seal it, and maintain a humid atmosphere while continuously producing and secreting antimicrobial molecules (Figure ).

Figure 1

Preparation and administration of live bacterial microparticles. Microparticles, formed by spray drying, can be administered directly onto open ulcers and lesions.

Results and Discussion

Microparticle Formation and Characterization

Microparticles containing B. subtilis were manufactured by spray-drying a bacterial suspension in PVA aqueous solution (2.5 wt %). Spray drying is a known method for the production of PVA microparticles in large quantities. The effect of B. subtilis on microparticle size and morphology was documented using dynamic light scattering (DLS) and scanning electron microscopy (SEM), respectively (Figure ).

Figure 2

Size and morphology: (A) Microparticle size distribution of microparticles with B. subtilis compared with empty microparticles as determined by DLS. (B) PVA microparticles and (C) B. subtilis encapsulated in PVA microparticles. Scale bar: 20 μm.

Empty microparticles showed an average diameter of 3.5 μm (Figure A), whereas B. subtilis microparticles exhibited a slightly larger diameter, around 4.5 μm. This increase in size can be attributed to the increase in viscosity of the PVA solution due to the presence of the bacteria.[20] Increasing the viscosity in the spray-drying process was found to increase droplet size distribution, which resulted in larger microparticles.[21] Regardless of the bacterial presence, microparticles larger than 1 μm in diameter tend to exhibit an anisotropic buckled morphology compared with the smaller microparticles, which were more spherical (Figure B,C). Studies on the formation of microparticles via spray drying have revealed that buckling is caused by the emergence of particle-dense regions at the surface of the droplet.[22] As the process of solvent evaporation progresses, the capillary forces exceed the electrostatic forces, causing deformation of the shell leading to nonspherical microparticles.[23] In other words, during the drying stage of the spray drying process, the interface of each droplet becomes solid-like and, as a result, the larger microparticles can only shrink further by crumbling of the outer shell.[24]

B. subtilis Analysis

To achieve possible effects of the spray-drying process on the fitness of bacteria, the growth rate of PVA-encapsulated bacillus was evaluated dynamically by a spectrophotometer (Figure A). By way of comparison, a similar experiment was carried out for equivalent amounts of both free, nonencapsulated bacteria and empty microparticles. Both groups reached a plateau about 8 h from the beginning of growth. However, while free bacteria started growing within 1 h, the encapsulated bacteria showed a lag of around 7 h. This “wake-up” period can be explained by several factors:[25] (1) extra time is required for rehydration and for the polymeric matrix to release the bacteria; (2) the environmental stress exposure during the spray-dry procedure may have decreased bacterial viability,[26] and (3) bacterial cells were exposed to both thermal and dehydration inactivation during spray drying, which may have inhibited bacterial growth. This delay in growth kinetics, previously documented for other bacteria strains,[27] could potentially be addressed by decreasing particle size or modifying process parameters. We note that Bacillus was identified before and after this assay with 99.92% accuracy (Crystal Gram-Positive ID Kit, BD, Maryland, USA). Thus, this in vitro assay supports our hypothesis that spray-dried B. subtilis may be used as a form of pharmaceutical dosage without compromising the viability, sterility, and shelf life of such formulations.

Figure 3

Characterization of B. subtilis. (A) Growth of B. subtilis in microparticles compared with fresh bacteria at 37 °C (n = 4). (B) LC/MS analysis of surfactin and fengycin produced and extracted from free bacteria. (C) LC/MS analysis of surfactin and fengycin produced and extracted from encapsulated bacteria.

Surfactin and Fengycin Extraction

The antimicrobial activity of B. subtilis is based on the bacteria’s ability to produce and secrete antimicrobial agents.[28] We therefore measured the release of surfactin and fengycin from free and from encapsulated B. subtilis (Figure B) by LC/MS (Figures S1–S3, Supporting Information). Surfactin production by B. subtilis started after 4 h, reaching a maximum of 350 μg/mL in 12 h. Fengycin production began after about 6 h, and its concentration reached a maximum of about 80 μg/mL in 12 h. Release of surfactin and fengycin from encapsulated B. subtilis showed comparable patterns with a 6 h delay, mirroring the delayed growth curve of the bacteria (Figure C). This manner in which surfactin is secreted before fengycin was attributed to the hemolytic and biofilm activities of the former.[29] In this regard, it is noteworthy that 200 μg/mL was found to be the minimum inhibitory concentration (MIC) for surfactin against S. aureus, while 250 μg/mL fully inhibited its growth.[30]

In Vitro Study

The antibacterial activity of B. subtilis microparticles against MRSA and S. aureus was examined using the disk-diffusion method. A lawn of each bacterium (100 μL of 108 CFU/mL) was spread on lysogeny broth (LB) agar Petri dishes using sterile beads followed by placement of microparticles (1 mg) at the center of each dish. After 48 h at 37 °C, the radius of inhibition was measured and compared with those of empty PVA microparticles (Figure A and Figure S4). The bacillus microparticles demonstrated antibacterial activity against both MRSA and S. aureus (inhibition zone diameters were 11 and 12 mm, respectively, p < 0.01), while empty PVA microparticles did not exhibit any activity. The activity of B. subtilis microparticles can be attributed, at least partially, to the secretion of a wide range of antibacterial molecules including surfactin and fengycin.[31] Similar experiments using pure surfactin, with concentrations between 100 and 400 μg/mL, were successful in inhibiting S. aureus and Escherichia coli (E. coli).[30,32] Surfactin is known to reduce and inhibit the formation of biofilm by S. aureus, causing an interruption in surface adhesion.[33]S. aureus, a human skin pathogen that causes a diverse range of serious hospital infections worldwide, has developed significant resistance against a variety of antibiotics.[34] Thus, the concept of administrating beneficial bacteria to environmental wounds can be seen as an alternative to antibiotics, including for the elimination of pathogenic bacteria such as S. aureus and MRSA.

Figure 4

(A) Antibacterial activity of B. subtilis encapsulated in microparticles, against methicillin-resistant S. aureus and S. aureus. Empty particles showed no zone of inhibition. (B) NIH 3T3 fibroblast viability 48 h after exposure to increased PVA microparticle concentration. * = statistically significant difference from all groups. P < 0.01 (n = 4).

We next evaluated the cytotoxicity of empty PVA microparticles on skin NIH 3T3 fibroblast cells using the cell viability assay (MTS) method (Figure B). Cells were seeded in wells (100,000 cells/well) and exposed to empty PVA microparticles at concentrations ranging between 0.625 and 2.5%, which is way below the maximum concentration allowed for PVA in cosmetics and skincare products.[35] Results show that cell survival is very high, around 90% (compared with nonexposed cells). Nevertheless, at a PVA concentration of 5%, a 30% cell death is observed, which is in agreement with another report.[36] Our results demonstrate well the potential of loaded PVA microparticles for treating skin wounds.

In Vivo Study

Next, tissue reaction to B. subtilis microparticles was determined using an open wound model in C57BL mice.[37] A single open wound was created by removing the skin (1 × 1 cm2) in the dorsal region of mice. Wounds were administered once daily with PVA microparticles, with or without B. subtilis. As a way of comparison, the dorsal region of mice of the same age and weight was also removed but left untreated. Lesion dimensions were measured, and wound area was documented daily by ImageJ. Mice were sacrificed after 5 or 15 days, and tissue from the injured area was harvested for histological analysis. Healing was evaluated by daily measuring wound size (Figure A,B) and by the toxic effect of the treatment as evaluated by histology (Figure C).

Figure 5

Wound closure in C57BL mice: (A) Representative photos showing the time course of wound healing. (B) Wound closure, normalized to the original size of the wound, expressed as mean ± SD. (C) Representative images of the in vivo response of skin to microparticles with and without B. subtilis after 5 and 15 days and the untreated group (scale bar = 1 mm). Red arrow - newly regenerated epidermal coverage; blue arrows - crust coverage of the wound site; green arrows - granulation tissue within the dermis and subcutis. *P < 0.05 versus empty and B. subtilis particles.

PVA microparticles (with and without B. subtilis) immediately attached themselves to the surface of the wound upon administration, remaining there until fully dissolved. Three days after injury, significant differences in wound closure were noticed between the two PVA-treated groups and the untreated group, with the former showing a reduction of 25% in wound size compared with the untreated group, which exhibited no difference or a slight increase in wound size. After 6 days, the wound area of the untreated group was around 95% of its initial size, compared with around 50% for the treated groups. Furthermore, the untreated wound was rougher and darker than the treated groups. These observations were also confirmed by histology, in which 5 days post-injury, a healthy healing process was evidenced by the formation of a crust and by the appearance of granulation tissue (marked by green arrows). This healthy process was verified when fresh epidermis tissue was created after 15 days.[38,39] After 12 days, both PVA groups showed complete re-epithelization and hair growth, compared with the untreated group, which required 15 days to heal.

The incorporation of B. subtilis into PVA microparticles, which demonstrated excellent in vitro performance, was relatively limited in the case of the in vivo healing process. A nonsignificant bacterial effect was noticed only during the first 9 days of the experiment, after which there was no apparent influence on the healing process. This discrepancy may be attributed to crust formation observed over the wound area 5 days after the beginning of the experiment (Figure C, marked by blue arrows). It is likely that the crust prevented bacteria and bacterial peptides from reaching the wounded area, thus limiting their efficacy to the first days of treatment. Further experimental studies are needed, however, to ascertain whether crust formation is indeed involved. Nevertheless, regardless of the bacterial presence, both PVA microparticle groups exhibited a faster healing rate than the untreated group, which is a prerequisite for decreasing the risk for early contamination.[40]

The histology data indicates no toxic effect related to PVA microparticles or to the B. subtilis incorporated in them. Data comparison between the three test groups indicates the presence of comparable changes, in both nature and severity. Moreover, the presence of B. subtilis did not delay healing of the induced wound, compared with the data evaluated at the 5 and 15 day time points. It can therefore be concluded that under the present experimental conditions, the use of B. subtilis incorporated into PVA microparticles in the wound healing model in mice is not associated with any adverse effects[41] and should be considered as safe.

Materials and Methods

Materials

PVA (Mw = 13,000–23,000 g/mol, 99% hydrolysis), glucose, surfactin, and fengycin were purchased from Sigma Aldrich (Sigma Chemicals, St. Louis, MO). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum were purchased from Gibco-Invitrogen Corp. (Grand Island, NY). Also used were Bacto Tryptone, Bacto Yeast extract, Bacto Agar, Bacto peptone (Becton Dickenson, NJ, USA), NaCl (Bio-Lab Chemicals, Jerusalem), MgSO4 (Spectrum Chemical, CA, USA), KH2PO4 (Riedel-de Haën, Munich), HCl 37% (Daejung Chemicals, Korea), and acetonitrile (J.T. Baker, NJ, USA). B. subtilis 3610 was generously provided by Prof. Ilana Kolodkin-Gal’s lab,[42] Weizmann Institute of Science, Israel. Bacterial cultures were American Type Culture Collection (ATCC) strains: S. aureus 25923 and methicillin-resistant 43300.

Microparticle Formation and Characterization

PVA microparticles containing B. subtilis were prepared using a Mini Spray Dryer B290 Advanced (B̈chi, Switzerland). PVA/bacterial solutions were prepared by suspending harvested bacteria pellets (2 × 109 CFU/mL) in sterile 2.5% aqueous PVA solution (80 mL). B. subtilis was incubated overnight at 37 °C and 200 rpm. The bacterial suspension was connected to the designated spray drier’s pump, which was adjusted to deliver 2.5 mL/min at an air flow of 600 L/h, with inlet and outlet temperatures set to 110 and 45 °C, respectively. Dried microparticles were collected from the cyclone and stored in a sterile Falcon tube at 4 °C. Control microparticles were also fabricated under similar conditions without bacteria. Microparticle morphology was characterized by SEM (SEM, Zeiss LEO 1530, Jena). Before imaging, samples were coated with 2.0 nm platinum using a sputter coater (208 HR Cressington, UK). The effect of the B. subtilis presence on microparticle size was measured using DLS (Malvern Panalytical, Cambridge, UK).

Bacterial isolates from microparticles were plated on LB agar Petri dishes and incubated for 24 h at 37 °C. A single colony was then transferred to a tube of inoculum fluid supplied with the BBL Crystal kit (Gram-Positive ID Kit-BD, Maryland, USA), and identifications were carried out according to the manufacturer’s instructions. To evaluate the concentration of B. subtilis in PVA microparticles, microparticles (2 mg) were suspended in 100 μL of LB medium. The suspension was plated on LB agar Petri dishes (n = 4) and incubated for 24 h, and colonies were counted using ImageJ software (NIH, Maryland, USA). As a way of comparison, control microparticles without bacteria were also evaluated. The possible effect of spray drying on the viability of B. subtilis was assessed by comparing the growth kinetics of encapsulated bacteria with those of a similar amount of free, nonencapsulated bacteria. Microparticles and free bacteria were cultured at 37 °C, and growth curves were dynamically monitored by a spectrophotometer (Synergy H1 Plate Reader Biotech Instruments Inc., Winooski, VT, USA) at λ = 600 nm over 24 h.

The antimicrobial properties of B. subtilis are based on the ability of the bacteria to produce and secrete antimicrobial agents. The release of antibacterial molecules by B. subtilis in microparticles was, therefore, monitored using encapsulated bacteria and was compared with that of free bacteria. Bacteria was diluted in 250 mL of an extract medium containing 20 g of glucose, 30 g of peptone, 7 g of yeast extract, 1.9 g of KH2PO4, and 0.45 g of MgSO4 in 1 L of DDW. The bacterial medium was incubated at 37 °C at 200 rpm. The bacterial medium (50 mL) was centrifuged at 8000g, at 4 °C for 10 min. The supernatant was adjusted to pH 2 with 6 M HCl, stored at 4 °C overnight, and centrifuged at 11,000g, for 20 min at 4 °C. The pellet was redissolved in DDW and lyophilized, and samples were redissolved in acetonitrile/DDW (80:20) and analyzed by LC/MS. Analysis was performed on a Waters UPLC H-class system equipped with a Waters Acquity C18 column (50 × 2.1 mm, 2.6 μm particle; injection volume 7 μL). The mobile phase consisted of solvent A (DDW containing 0.1% trifluoroacetic acid) and solvent B (acetonitrile containing 0.1% trifluoroacetic acid). The following linear gradient elution was used: 50% A at 0 min, decreased to 3% A from 0 to 10 min, held at 3% A from 10 to 12 min, then increased to 50% A from 12 to 12.5 min, and further held at 50% A until 17 min. The flow rate was set at 0.4 mL/min, and effluent absorbance was monitored at 210 nm. Mass spectrometry was performed using a Waters Xevo G2 QTof system operating in electrospray ionization positive-ion mode. Cone and probe capillary voltages were 45 V and 3.0 kV, respectively. Source and desolvation temperatures were 120 and 400 °C (desolvation gas flow (N2), 800 L/h, cone gas flow, 1 L/h). Results were compared with those obtained from commercial surfactin and fengycin.

The antibacterial activity of the PVA microparticles was determined against S. aureus and MRSA using the disk diffusion method.[43] In brief, a lawn of each bacterium (100 μL of 108 CFU/mL) was spread on LB agar Petri dishes using sterile beads. Microparticles (1 mg) were placed at the center of each dish using a sterile spatula. Petri dishes were incubated for 48 h at 37 °C, and the radius of inhibition was measured. As a way of comparison, the activity of empty microparticles was also measured using the same method.

Microparticle cytotoxicity was evaluated by exposing NIH 3T3 fibroblast cell lines to increased concentrations of PVA microparticles. Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum. Cultures were maintained at 37 °C in a 95% air/5% carbon dioxide atmosphere, at 95% relative humidity. Cytotoxicity was assessed after 24 h by the MTS assay using CellTiter 96 solution according to the manufacturer’s instructions. Results are expressed relative to unexposed cells (n = 4).

In Vivo Study

Animals were cared for in compliance with protocols approved by the Council for Animal Experiments, Israel Ministry of Health, in conformity with the Animal Welfare law guidelines (published in 1994). A total of 21 eight-week-old C57BL mice (Envigo, Jerusalem, Israel) were anesthetized with 1% isoflurane (Piramal Critical Care, Inc. PA, USA) in oxygen. The dorsal region was removed, creating an open wound (1 × 1 cm2). Mice were randomly assigned to one of the following three groups: empty PVA microparticles, PVA microparticles containing B. subtilis, and a control group that did not receive any treatment (“no treatment”). Each treatment was administered daily for the entire period of the experiment, and lesion dimensions were measured using ImageJ software (NIH, Maryland, USA). Mice were sacrificed after 5 or 15 days, and dorsal skin samples were harvested and kept in 10% formalin for histology analysis. Skin samples were stained with hematoxylin and eosin and Masson’s trichrome for histology evaluation, which was performed by a board-certified toxicologic pathologist (AN).

Statistical Analysis

Results of surfactant extraction, inhibition rates, cytotoxicity MTS assays, and in vivo studies are presented as mean values ± SD. Statistical comparisons were performed using Prism 5, GraphPad (San Diego, CA). The t test was used to analyze the significance of the differences between the treated groups; p values <0.01 were considered to reflect statistical significance (n = 4).

Conclusions

The pharmacological activity of PVA microparticles was successfully demonstrated in an open wound model. Microencapsulation techniques for live bacteria include extrusion, emulsification, freeze-drying, and spray drying.[44] The delivery system described here, which is based on spray drying, was chosen based on its advantages over the other techniques: microspheres formed by extrusion tend to be less stable and do not lend themselves to large-scale production.[45] Emulsification involves organic solvents, often toxic to the bacteria; although freeze drying is considered to be very efficient, it is, however, limited by very high production costs.[46] Thus, spray drying is the most widely used technique for microencapsulation of live bacteria, being relatively simple and cost effective, and amenable to large-scale production. Microparticles were produced by one simple step that is amenable to large-scale production. Wounds treated with PVA microparticles were closed and healed after 12 days, compared with 15 days for the nontreated groups. There was no evidence of skin irritation, infection, or other adverse effects during the 15 day postoperative observation period. In vitro, the bacillus microparticles demonstrated remarkable antibacterial activity against MRSA and S. aureus, but in this study, the bacterial effect was limited to the first days of the in vivo study, probably due to crust formation on the wound surface. Nevertheless, it was shown that the use of live bacterial formulations has the potential to continuously produce antimicrobial agents and inhibit the growth of pathogenic bacteria. Since B. subtilis peptides were proven to be therapeutic agents for open wounds and skin disorders,[47,48] a live bacterial delivery approach that continuously secrete antimicrobial peptides could open the door to a new class of delivery systems for biological molecules, with potential in many acute and chronic skin diseases. PVA microparticles may be administered as compact (pressed) powder using a fluffy sponge or a brush, dispersed in liquid oils (e.g., paraffin or vegetable oil), or applied using a powder blower or microneedles.[49]