Drug Delivery
.jpg "Yang Wu, PhD (he/him/his) photo")
Yang Wu, PhD (he/him/his)
Postdoctoral Research Associate
University of Nebraska Medical Center
Omaha, Nebraska, United States
Siwei Zhao
Assistant Professor
University of Nebraska Medical Center, United States
Biofilm infection can lead to chronic, non-healing wounds (CWs). The current clinical care for CW biofilm infection uses debridement and antibiotic treatment. Debridement removes biofilm-contaminated tissue and reduces bacterial load. However, biofilm can quickly recover after debridement, so repeated debridement is required. Antibiotics administrated via systemic or topical routes have a low bioavailability in wound tissue, necessitating long-term or high-frequency dosing to maintain a low bacterial count in CWs. The current treatment imposes a heavy burden on both patients and our healthcare system.
Nanoparticle-based antibiotic formulations allow sustained drug release and can potentially reduce antibiotic dosing frequency. However, topical application, the most common route of nanoparticle administration, has a low delivery efficiency due to the slow diffusion process, leading to unsatisfactory biofilm inhibition. There is a critical need for a high-efficiency nanoparticle delivery technology that can rapidly deliver a therapeutic dose of antibiotic nanoparticles into CWs to achieve a long-term inhibition of biofilm infection.
PLGA/Van was synthesized using double-emulsion methods. 40 mg Resomer-RG653H was dissolved in 2 mL dichloromethane and emulsified with 0.5 mL 1% vancomycin solution on ice using probe ultrasonicator. Primary emulsion was then mixed with 10 mL solution containing 1% PVA and 0.01% chitosan and emulsified again. Secondary emulsion was subsequently stirred for 6 hours. Nanoparticles were isolated and washed in 50kDa cut-off centrifugal filters. For unloaded PLGA nanoparticles, 0.5 mL H2O was used instead in primary emulsion.
Vancomycin release was monitored using Slide-A-Lyzer™ Dialysis Devices. 5 mg PLGA/Van was dialyzed against MilliQ-water at 37oC, from which samples were withdrawn periodically for UV-Vis measurements. Same volume of MilliQ-water was supplemented afterwards.
HIC-based iontophoresis device was designed according to our published paper[1]. The device was fabricated with laser micromachining and acrylic plastics, and assembled using VHB tape. Polyethylene glycol (PEG) hydrogel (10% PEGDMA MW8000, 5% PEGDA MW700, and 1% Irgacure-2959) was fabricated with UV crosslinking and was attached to the device using benzophenone-assisted bonding. The anode solution contains 0.6M Na2HPO4, and the cathode solution contains 0.6M NaH2PO4 and 0.48M Na2HPO4. Carbon rods were used to supply electric current to our device.
Iontophoretic nanoparticle delivery was conducted on 6-mm excisional porcine skin wounds and quantified via UV-Vis measurements after extracting the treated biopsy in 2% acetic acid and acetonitrile. Delivered nanoparticles were allowed to release vancomycin for 3 days before wounds were inoculated with Methicillin-resistant Staphylococcus aureus (MRSA) at 108 CFU/mL. Wounds were then incubated at 37oC for 2 days before bacterial quantification.
Both PLGA/Van and unloaded PLGA nanoparticles had an average size of ~300 nm, were monodispersed (PDI< 0.3) and positively charged (zeta potential >30mV). DL% and EE% (Figure 1) were consistent with what others have reported[2][3]. Vancomycin release was continuously monitored for 26 days. It shows that vancomycin released fast in the first 3 days. In total 9.5 μg vancomycin per mg of PLGA/Van was released by Day 3 (Figure 2). This increased to 10.8 μg/mg on Day 5, which accounted for 32.7% of vancomycin loaded in PLGA/Van. The release didn’t increase significantly from Day 5 to 26.
PLGA/Van delivery was performed using our HIC-based iontophoresis system. 20 mg PLGA/Van dispersed in PBS was loaded in drug chamber. A high current density of 75 mA/cm2 was used to deliver PLGA/Van into ex vivo porcine skin wound for 1 hour. A conventional low-intensity iontophoresis at 0.5 mA/cm2 and passive diffusion were also tested as comparisons. 240.2 µg/g vancomycin (7.21 mg/g PLGA/Van) was delivered into wound tissue using 75 mA/cm2 iontophoresis, while only 26.2 and 16.3 µg/g vancomycin (0.79 and 0.49 mg/g PLGA/Van) were delivered by 0.5 mA/cm2 iontophoresis and passive diffusion (Figure 3). Treated wounds were further challenged with MRSA at 3-day post-delivery to evaluate biofilm inhibition efficacy. 108.30 CFU/g biofilm was harvested from untreated wounds at Day 2 post-inoculation, similar to those treated with unloaded PLGA nanoparticles (108.02 and 108.21 CFU/g) (Figure 4). PLGA/Van delivered by passive diffusion and 0.5 mA/cm2 iontophoresis achieved low bacterial reductions of 0.62 and 1.43 Log10-scales respectively, compared with untreated wounds. However, PLGA/Van delivered by 75 mA/cm2 iontophoresis achieved a significantly higher biofilm inhibition efficacy. The bacterial count was measured to be 104.70 CFU/g at Day 3 (3.61 Log10-scales lower than untreated control), which was below clinical threshold for wound infections.
In conclusion, we demonstrated that our HIC-based high-intensity iontophoresis technology significantly enhanced antibiotic nanoparticle delivery efficiency into ex vivo porcine skin wounds and achieved better biofilm inhibition efficacy. In the future, we plan to test biofilm inhibition efficacy using an in vivo wound infection model and determine how our treatment affects the wound healing process.