Recipient Organization
COLORADO STATE UNIVERSITY
(N/A)
FORT COLLINS,CO 80523
Performing Department
Microbiology, Immunology and Pathology
Non Technical Summary
Bacteria are becoming more and more resistant to antibiotics, so we need to develop other ways to treat infections. We are developing methods to treat bacterial infections using genetically altered enzymes that will breakdown the cell wall of bacteria and kill them. These antibacterial enzymes will be incorporated into tiny particles and embedded in wound dressings. In addition to being antibacterial, the wound dressings will change colors if bacteria begin to grown in the wound. The wound dressings will be used to treat animals that have wound infections so we will not have to rely on antibiotics for treatment.
Animal Health Component
80%
Research Effort Categories
Basic
10%
Applied
80%
Developmental
10%
Goals / Objectives
Specific Aim 1: Develop micelles and nanoparticles for delivery of bacteriolytic and biofilm degrading enzymes to the wound site.Specific Aim 2: Evaluate the antibacterial nanofiber wound dressing containing bacteriolytic/biofilm disrupting enzymes for activity.
Project Methods
Fabrication of micelles containing bacteriolytic and antibiofilm enzymes. Pluronic® 65 will be used to encapsulate lytic enzymes in micelles. Micelles will be filtered with a 0.8-µm pore size filter and particle size will be measured with a Zeta Sizer. Fabrication of gentamicin and mupirocin (Gm and Mup) - loaded nanoparticles. Poly(lactide-co-glycolide) (PLGA, RESOMER® biodegradable polymers) will be used to encapsulate these antibiotics. (1) Gm or Mup (1:1) will be dissolved in water and emulsified with dichloromethane containing PLGA. This emulsion will be emulsified with polyvinyl alcohol (PVA) at pH 7.4. (2) Gm or Mup (1:1) will be dissolved in water and added to acetone containing PLGA. The diffusion of water into the acetone will result in the formation of nanoparticles. The s/o phase will then be added to PVA buffer and diffusion of the acetone phase into the aqueous PVA phase will result in the coating of Gm with PLGA The resulting NPs will be used without any further purification. Particles will be characterized by particle size and zeta-potential (surface charge) using TEM and a Malvern Zeta Sizer, respectively. Particle chemistry will be evaluated by X-ray photoelectron spectroscopy, and FT-IR spectroscopy. 'All-in-one' nanofiber composite (NFC) fabrication. The Gm- and Mup-PLGA NPs and lytic enzyme micelles will be incorporated into the PU-PDA nanofiber to make the NFCs. PU-PDA nanofiber will be treated using a plasma surface treatment method, to improve wettability and increase active/functional groups. Gm- and Mup-PLGA NPs and lytic enzyme micelles will be prepared at a concentration of 1, 5, and 10 % by weight. The PU-PDA fiber will be added to the suspension, incubated, and then NFCs will be taken out, rinsed with DI water, and air-dried, resulting in the NFCs coated with Gm/Mup PLGA NPs and lytic enzyme micelles. The coating microstructures and strength of the Gm- and Mup-PLGA NPs and lytic enzyme micelles on the NFCs will be examined using scanning electron microscopy and atomic force microscopy. Evaluation of biosensor sensitivity and specificity. Biosensor sensitivity for colorimetric detection will be performed by preparing serial dilutions of log phase P. aeruginosa, adding the bacteria onto the nanofibers obtained, and measuring color reflectance (R) over time using a ColorQuest spectrophotometer. These experiments will be performed in triplicate. Uninoculated growth media and log phase P. aeruginosa will serve as the negative and positive control, respectively. The means of each measurement will be calculated. The specificity of biosensor detection and the exhibition of a specific wavelength for each bacterium (wavelength 'fingerprint') will be tested using the ColorQuest spectrophotometer. To determine if the wavelength is associated with a specific bacteria, the fiber will be tested using various clinically relevant bacteria. Evaluation of enzyme activity and stability. The activity of the purified chimeric enzymes will be measured in 8 different formulations: 1) purified enzymes PelAh, PslGh with Art-175 (hereafter referred to as the PPA enzyme complex), 2) purified enzymes PelAh, PslGh with Jel-RC (hereafter designated the PPJ enzyme complex), 3) the PPA enzyme complex in poloxamer micelles, 4) the PPJ enzyme complex in poloxamer micelles, 5) nanofibers coated with micelles containing PPA enzyme complex, 6) nanofibers coated with micelles containing PPJ enzyme complex, 7) nanofibers coated with micelles containing a combination of PPA enzyme complex and antibiotics (Gm and Mup), and 8) nanofibers coated with micelles containing a combination of PPJ enzyme complex and antibiotics (Gm and Mup). Stability of the enzyme in each formulation will be tested as described previously. The turbidity plate lysis assay will be used to determine the specific activity of each of these preparations P. aeruginosa PAO1 as described previously. Controls will include buffer, micelles lacking enzymes, and pristine nanofibers. Each formulation will be assayed in triplicate and the mean values will be subjected to statistical analysis. Evaluation of the controlled release of gentamicin and mupirocin from nanoparticles. Release of Gm and Mup from the nanoparticles will be measured as described previously. For Gm, the nanoparticles (10 mg/ml PLGA) will be placed into a dialysis chamber with a 10,000 Da membrane; dialysate will be collected at different times, diluted with boric acid, and derivatized with orthophthaldehyde. By measuring the ratio of fluorescence (360/460), and comparing with a standard curve of fluorescent derivatized Gm, the amount of Gm released by the nanoparticles can be determined. Release of Mup will be measured by collecting dialysate and subjecting it to HPLC as described previously. Evaluation of biofilm destruction by lytic enzyme. Degradation of P. aeruginosa biofilms will be tested by growing fluorescent-labeled P. aeruginosa PAO1 (obtained from Dr. Brad Borlee, CSU) in a Drip Flow Biofilm Reactor. Biofilm coupons will be exposed to: 1) the PPA enzyme complex, 2) the PPJ enzyme complex, 3) the PPA enzyme complex in poloxamer micelles, 4) the PPA enzyme complex in poloxamer micelles, 5) nanofibers coated with micelles containing PPA enzyme complex, 6) nanofibers coated with micelles containing PPJ enzyme complex, 7) nanofibers coated with micelles containing a combination of PPA enzyme complex and antibiotics, and 8) nanofibers coated with micelles containing a combination of PPJ enzyme complex and antibiotics. Biofilm reduction will be monitored by confocal microscopy and viable bacterial counts as described in ASTM E2647-13 Standard Test Method for Quantification of P. aeruginosa Biofilm Grown Using Drip Flow Biofilm Reactor with Low Shear and Continuous Flow. Controls will consist of biofilms exposed to enzyme buffer solution and the pristine nanofibers. Confocal laser-scanning microscopy (Zeiss, LSM510 META) of the biofilms post-treatment will be performed as described previously.The number of P. aeruginosa cells attached to the coupon, surface coverage, average thickness, maximum thickness, roughness coefficient, and surface-to-volume ratio of the biofilms will be measured using COMSTAT . Three images will be taken in duplicate for each coupon in three independent experiments. A modified Kirby-Bauer agar disk diffusion assay will also be used to compare the ability of different formulations to inhibit bacterial growth. These experiments will be performed in triplicate on three different plates and the mean values will be recorded and subjected to statistical analysis.Statistical analysis will be performed in consultation with Dr. Ann Hess (Statistics Department, CSU). ANOVA will be used to determine if there is a statistically significant difference in the color change of the fiber when exposed to P. aeruginosa and clinically relevant bacteria. In these experiments the means of the ratios of the wavelengths measured after exposure of the fiber to bacteria will be compared to that following exposure to a buffer control and to intact P. aeruginosa. ANOVA will also be used to determine if there is a statistically significant difference in the ability to inhibit P. aeruginosa in the Kirby Bauer assay and to reduce P. aeruginosa biofilms as compared to untreated controls when measured by zones of inhibition, viable counts, and confocal microscopy. All experiments will be performed in triplicate with 3 separate fiber samples/micelles/nanoparticles produced on 3 different occasions. Tukey or Dunnett pairwise comparisons will be used where appropriate.