Ultrasound-Mediated Release of Antibiotics from Polymers for Surgical Site Infections

Introduction: Most surgical implants are made from either non-degradable metals or polymers that can only be removed surgically. This can lead to repeat surgeries, repeat hospital visits, inflammation, and infection. Two common bacteria found in surgical site infections are Staphylococcus Epidermidis and Escherichia Coli. These bacteria cause infections that if untreated, could result in hospitalization and death. One current method for infection treatment in surgical sites includes intramedullary and subcutaneous perfusion with large doses of antibiotics. As an alternative to surgical removal and antibiotic perfusion, we have developed ultrasound-responsive polymers capable of being degraded on-demand by high-intensity focused ultrasound (HIFU) for the release of entrapped antibiotics. We chose to explore inhibition and knockdown of these bacteria strains with release of clinically relevant antibiotics Vancomycin and Gentamicin.

Polycaprolactone (PCL) polymers were crosslinked with reversible Diels-Alder (DA) linkers that underwent DA retrograde-reaction when exposed to HIFU, which induced energy transfer within the material. The use of HIFU as a non-invasive external trigger resulted in image-guided and spatiotemporally controlled degradation of PCL and the resulting release of antibiotics. Constructs were evaluated on mechanical properties, degradation, biocompatibility, and release kinetics for differing focused ultrasound parameters. The released antibiotics were compared to stock solutions at the same concentration in order to compare activity against both planktonic and biofilm based in vitro bacteria colonies. The in vivo evaluation of the bacteria knockdown is to be completed with a subcutaneous pocket model containing the antibiotic-loaded polymer construct and 1 million colony forming units of the bacteria strains.

Materials and

Methods: PCL FDA represents PCL that leverages furan-based Diels-Alder cycloaddition for thermo-responsive crosslinking. PCL diol is reacted with methylene diphenyl diisocyanate to create a PCL cyanate, this intermediate is then reacted with furfuryl amine. Bismaleimide was added to form the furan-based Diels-Alder construct referred to as PCL FDA. Isosorbide was used to crosslink PCL cyanate without a thermo-responsive linker, resulting in the control group PCL ISO. During final crosslinking steps, antibiotics were added to the solution creating a polymer matrix entrapping Vancomycin, Gentamicin, or the combination of both (Figure 1A).

Following the creation of the polymer systems, we wanted to fully characterize PCL FDA and PCL ISO. All polymer products were verified using HNMR and FTIR and characterized using DSC. Biocompatibility of the biomaterials and degradation byproducts were verified by mass spectrometry and assessed for viability with representative LIVE/DEAD, metabolic rate through CellTiter blue, and cell count through PicoGreen. Retention of payloads and mass loss were assessed at in vivo parameters via immersion bath. Optical profilometry, tensile testing, and imaging (Figure 1B) were used to further understand polymer change from ultrasound exposure.

For the main application of the polymer constructs, we tested ultrasound-stimulated release of antibiotics for bacteria knockdown and inhibition. We assessed the release kinetics of model payloads and antibiotics via focused ultrasound stimulation at varying ultrasound parameters. The released antibiotics and their activity compared to stock antibiotic solutions were assessed in an in vitro planktonic culture of Escherichia Coli, Staphylococcus Epidermidis, and a coculture of both bacteria strains.

Results, Conclusions, and Discussions: Results and

Discussion:
Following the verification of the polymer synthesis and trends of the thermo-responsive crosslinking, polymers were proven to be biocompatible both as a construct and as degradation byproducts through representative LIVE/DEAD, metabolic rate, and cell proliferation. HIFU successfully triggered the DA retro-reaction causing higher levels of degradation and antibiotic release (Figure 1C) in PCL-FDA compared to PCL-ISO. PCL-FDA underwent larger changes in surface topography, mechanical properties, mass loss, and antibiotic retention following ultrasound exposure when compared to PCL-ISO. Antibiotics released via focused ultrasound stimulation from PCL-FDA were compared to stock antibiotic solutions for successful knockdown and inhibition of bacteria with no significant decrease in activity via release (Figure 1D).

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Figure 1. (A) Graphical depiction of the Diels-Alder retrograde-reaction triggered by HIFU and the associated antibiotic release. (B) Representative image of PCL-FDA before and after 15 minutes of focused ultrasound exposure at 1.1 MHz with a peak positive pressure of p+ = 75 MPa and peak negative pressure of p– = 15 MPa. (C) Gentamicin, Vancomycin, and combinatorial release from PCL-FDA and PCL-ISO using a 3.68 MHz focused ultrasound transducer with a peak positive pressure of p+ = 78 MPa and peak negative pressure of p– = 21 MPa for 3 and 5 minutes of exposure (n = 4, ns = no significance, **** = p < 0.0001). (D) Knockdown of Escherichia Coli, Staphylococcus Epidermidis, and a coculture of both bacteria via released antibiotics from PCL-FDA compared to stock solutions to ensure no statistically significant decrease in antibiotic activity (n = 6).

Conclusions:
PCL crosslinked with stimuli-responsive DA linkers successfully released antibiotics at higher rates than samples crosslinked without a responsive crosslinker. HIFU proved to be a successful external trigger to spatiotemporally control the degradation of polymer matrices and the release of antibiotics. Antibiotics released from Diels-Alder crosslinked polymer matrices proved to have no significant decrease in activity and proved effective in inhibition and knockdown of clinically relevant bacteria strains in vitro. Ongoing work for the upcoming summer includes an in vivo evaluation of bacteria knockdown via ultrasound stimulation of the antibiotic-loaded polymer matrix.

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