Wearable adjunct ozone and antibiotic therapy system for treatment of Gram-negative dermal bacterial infection

Abstract

The problematic combination of a rising prevalence of skin and soft tissue infections and the growing rate of life-threatening antibiotic resistant infections presents an urgent, unmet need for the healthcare industry. These evolutionary resistances originate from mutations in the bacterial cell walls which prevent effective diffusion of antibiotics. Gram-negative bacteria are of special consideration due to the natural resistance to many common antibiotics due to the unique bilayer structure of the cell wall. The system developed here provides one solution to this problem through a wearable therapy that delivers and utilizes gaseous ozone as an adjunct therapy with topical antibiotics through a novel dressing with drug-eluting nanofibers (NFs). This technology drastically increases the sensitivity of Gram-negative bacteria to common antibiotics by using oxidative ozone to bypass resistances created by the bacterial cell wall. To enable simple and effective application of adjunct therapy, ozone delivery and topical antibiotics have been integrated into a single application patch. The drug delivery NFs are generated via electrospinning in a fast-dissolve PVA mat without inducing decreasing gas permeability of the dressing. A systematic study found ozone generation at 4 mg/h provided optimal ozone levels for high antimicrobial performance with minimal cytotoxicity. This ozone treatment was used with adjunct therapy delivered by the system in vitro. Results showed complete eradication of Gram-negative bacteria with ozone and antibiotics typically used only for Gram-positive bacteria, which showed the strength of ozone as an enabling adjunct treatment option to sensitize bacteria strains to otherwise ineffective antibiotics. Furthermore, the treatment is shown through biocompatibility testing to exhibit no cytotoxic effect on human fibroblast cells.

Figure 1

Wearable adjunct ozone and topical antibiotic therapy system. (a) Ozone and antibiotic adjunct therapy can be used as an alternative treatment for skin and soft tissue infections that do not respond to traditional therapies. Ozone provides antimicrobial properties and enables antibiotic to enter cell and disrupt cell functions, such as protein production. (b)The system utilizes gaseous ozone and gas permeable and drug-eluting nanofiber mat for treating developing wounds in the following process: (i) All-in-one wound patch with drug-eluting nanofiber mesh and gas permeable membrane is applied to skin wound. (ii) NFs begin to dissolve and release the topical antibiotics. (iii) Ozone is applied to the system for full treatment duration as topical antibiotics are completely released from NFs. Ozone and antibiotics work together to eliminate infection. (iv) Once the wound has healed, the wound patch is removed from the area. Combination of ozone and antibiotic treatment can treat antibiotic resistance infections and prevent development of new infections, leading to faster healing times.

Figure 2

Portable adjunct ozone and topical antibiotic therapy system. (a) Ozone wound treatment system designed to administer adjunct ozone and antibiotic therapy topically to dermal wounds. System comprised of portable ozone generation system with microblower for ozone delivery and a porous mesh of drug-eluting PVA nanofibers for antibiotic delivery. Portable rechargeable system is fitted to custom housing and utilizes onboard low-power electronics for. (b) Relationship of concentration of ozone created by portable system to the mass generation rate. Error bars denote standard deviation.

Figure 3

Properties of electrospun NF mat. Microscope image of (a,e) ozone delivery patch surface and (b,f) after coating with linezolid containing NFs, (c,g) and after coating with vancomycin containing NFs, (d,h) after the dissolution of the NFs. Images were taken using optical microscope (a–d) and SEM (e–h). (i) Histogram displaying frequency of pore size within vancomycin and linezolid spun fiber mats. (j) Contact angle measurement of dressing at various stages of treatment. Error bars denote standard deviation.

Figure 4

Porosity characterization of ozone dressing with and without drug eluting NFs coating. (a) Internal flow resistance at varying flowrates for dressing at different stages of application. (b) Comparison of internal flow resistance at 25 mL/min. Error bars denote standard deviation.

Figure 5

Dissolution characterization of drug eluting NFs. (a) Dissolution rate of NF fabricated with partially hydrolyzed and fully hydrolyzed PVA. (b) Dissolution over time of partially hydrolyzed NFs infused with drug mimicking dies (Red for vancomycin and Blue for linezolid). (c) Proportion of material dissolved by critical time of 10 min (< 3% total treatment duration) for NFs in liquid and gel media. (d) Comparison of time needed to achieve critical dissolution of blue dye (linezolid) NFs in buffer solution with different pH levels. Error bars denote standard deviation.

Figure 6

Antimicrobial efficacy and cell viability under continuous exposure to differing levels of ozone (2–8 mg/h). Antimicrobial effect against (a) P. aeruginosa and (b) E. coli bacteria cultures in PBS over the course of 8 h. (c) Cell viability of human fibroblasts treated with 6 h of ozone at 2–8 mg/h at 37 °C. Viability was measured 1 day, 3 days, and 7 days after treatment ended. (d) Live/Dead staining of human fibroblast cells exposed to varying levels of ozone therapy 1 day and 7 days after treatment. Error bars denote standard deviation.

Figure 7

Antibacterial efficacy against bacteria cultures in TSB media and cell viability results of adjunct ozone and antibiotic therapy test in vitro at 37 °C. (a) Results for ozone + linezolid and ozone + vancomycin adjunct therapy on P. aeruginosa. (b) Antibacterial results of ozone + linezolid and ozone + vancomycin adjunct therapy on E. coli. Ozone was applied at 4 mg/h for 6 h. Linezolid and vancomycin were applied in solution at 200 μg/mL. (c) Viability of human fibroblast cells exposed to 6 h of ozone, ozone + vancomycin, and ozone + linezolid treatment measured 1 day, 3 days and 7 days after treatment ended. (d) Live/Dead staining of human fibroblasts 1 day and 7 days after treatment ended. Error bars denote standard deviation.