Controlled Delivery of Vancomycin from Collagen-tethered Peptide Vehicles for the Treatment of Wound Infections

Abstract

Despite the great promise of antibiotic therapy in wound infections, antibiotic resistance stemming from frequent dosing diminishes drug efficacy and contributes to recurrent infection. To identify improvements in antibiotic therapies, new antibiotic delivery systems that maximize pharmacological activity and minimize side effects are needed. In this study, we developed elastin-like peptide and collagen-like peptide nanovesicles (ECnVs) tethered to collagen-containing matrices to control vancomycin delivery and provide extended antibacterial effects against methicillin-resistant Staphylococcus aureus (MRSA). We observed that ECnVs showed enhanced entrapment efficacy of vancomycin by 3-fold as compared to liposome formulations. Additionally, ECnVs enabled the controlled release of vancomycin at a constant rate with zero-order kinetics, whereas liposomes exhibited first-order release kinetics. Moreover, ECnVs could be retained on both collagen-fibrin (co-gel) matrices and collagen-only matrices, with differential retention on the two biomaterials resulting in different local concentrations of released vancomycin. Overall, the biphasic release profiles of vancomycin from ECnVs/co-gel and ECnVs/collagen more effectively inhibited the growth of MRSA for 18 and 24 h, respectively, even after repeated bacterial inoculation, as compared to matrices containing free vancomycin, which just delayed the growth of MRSA. Thus, this newly developed antibiotic delivery system exhibited distinct advantages for controlled vancomycin delivery and prolonged antibacterial activity relevant to the treatment of wound infections.

Keywords: collagen; collagen-like peptide (CLP); elastin-like peptide (ELP); hydrogel; methicillin-resistant Staphylococcus aureus (MRSA); vancomycin; wound dressing.

Figure 1.

Characterization of vancomycin loaded ECnVs (A) Size measurement of ECnVs using DLS before and after vancomycin loading and after lyophilization. (B) Representative TEM images of ELP-CLP before and after Vancomycin loading. Scale bar is 500 nm.

Figure 2.

Vancomycin release from nanocarriers (A) without removing or (B) with mathematically removing the unloaded Vancomycin at 37 °C. The release profiles were fitted with Korsmeyer-Peppas model. (C) Table for encapsulation efficiency (EE) and loading capacity (LC) of vancomycin in liposome and ECnVs and constants for Korsmeyer-Peppas model fitting. K_kp indicates Korsmeyer release rate constant and n indicates diffusional exponent. Each data represents mean ± standard deviation for n=6.

Figure 3.

ECnVs retention on matrices at 37 °C. (A) Representative 3D plotted confocal images of ECnVs-Cy3 (Red) and collagen (Grey) in Collagen matrix or Co-gel matrix before and after wash. 1 unit is 25 μm. (B) Image quantification for fluorescent intensity of ECnVs after wash normalized to the intensity before wash. Each data represents mean ± standard deviation for n=6. An unpaired student’s t-test with equal variance was used to detect statistical significance. *p<0.0001 for co-gel relative to collagen.

Figure 4.

ECnVs release from matrices at 37 °C for 96 h. Release profile were fitted Kormeyer-Peppas model. Each data represents mean ± standard deviation for n=3. The statistical difference of K_kp states *p<0.05.

Figure 5.

Vancomycin release from ECnVs tethered in (A) collagen or (B) co-gel matrix. (C) Release profile were fitted by first-order kinetics from 0 to 8 h (solid line) and Kormeyer-Peppas model from 8 h to 96 h (dotted line). K_i = first order constant, K_kp = Korsmeyer release rate constant, and n = diffusional exponent. Each data represents mean ± standard deviation for n=4. The statistical difference of K_i states *p<0.05.

Figure 6.

Anti-bacterial activity of vancomycin loaded ECnVs tethered collagen/co-gel matrices against MRS A . Optical density measurement of MRS A cultures grown in blank collagen/co-gel (black circle), vancomycin (3 μg/mL) loaded collagen/co-gel (grey square), and vancomycin (3 μg/mL) loaded ECnVs tethered collagen/c-gel (white triangle) with a total of two bacterial inoculations (16 h per inoculation). Each data represents mean ± standard deviation for n = 3.