Polyarginine Decorated Polydopamine Nanoparticles With Antimicrobial Properties for Functionalization of Hydrogels

Abstract

Polydopamine (PDA) nanoparticles are versatile structures that can be stabilized with proteins. In this study, we have demonstrated the feasibility of developing PDA/polypeptides complexes in the shape of nanoparticles. The polypeptide can also render the nanoparticle functional. Herein, we have developed antimicrobial nanoparticles with a narrow size distribution by decorating the polydopamine particles with a chain-length controlled antimicrobial agent Polyarginine (PAR). The obtained particles were 3.9 ± 1.7 nm in diameter and were not cytotoxic at 1:20 dilution and above. PAR-decorated nanoparticles have exhibited a strong antimicrobial activity against S. aureus, one of the most common pathogen involved in implant infections. The minimum inhibitory concentration is 5 times less than the cytotoxicity levels. Then, PAR-decorated nanoparticles have been incorporated into gelatin hydrogels used as a model of tissue engineering scaffolds. These nanoparticles have given hydrogels strong antimicrobial properties without affecting their stability and biocompatibility while improving their mechanical properties (modulus of increased storage). Decorated polydopamine nanoparticles can be a versatile tool for the functionalization of hydrogels in regenerative medicine applications by providing bioactive properties.

Keywords: antimicrobial properties; hydrogel; nanoparticles; polyarginine; polydopamine.

FIGURE 1

Different steps to synthesize PDAPAR₃₀ NPs.

FIGURE 2

(A) Picture showing the stability after 2 months at 4°C of the different PDA-PAR₃₀ NPs formulations using different concentrations of dopamine hydrochloride (0.5, 0.3, and 0.2 mg.mL^–1) with a defined concentration of polyarginine (PAR) of 1 mg.mL^–1. (B) Typical image of PDA-PAR₃₀ 0.2 NPs obtained with Transmission Electron Microscope (TEM) and the subsequent size quantification results. (C) Results of zeta potential measurements performed on PDA-PAR₃₀ 0.2 NPs as compared to pure PDA particles.

FIGURE 3

(A) Normalized S. aureus growth in supernatant after 24 h in contact with different PDA-PAR₃₀ 0.2 NPs at different dilutions to determine the Minimum Inhibitory Concentration (MIC) (n = 3 and error bars correspond to standard deviations) (***p < 0.001). (B) Cytotoxicity test of the same NPs dilutions after 24 h of exposure with Balb 3T3 cells (n = 3 and error bars correspond to standard deviations).

FIGURE 4

(A) Macroscopic pictures of the resulting Gelatin NPs hydrogel composite (Gel-PDA-PAR₃₀) compared to pure Gelatin hydrogel (Gel). (B) Scanning Electron Microscopy images of the Gel-PDA-PAR₃₀ hydrogel. (C) Swelling properties of the Gel-PDA-PAR₃₀ hydrogel compared to pure Gelatin hydrogel at different time points in PBS at 37°C (n = 3 error bars correspond to standard deviations). (D) Kinetic of PDA-PAR₃₀-FITC NPs (non-diluted) release from Gelatin hydrogel at 37°C in PBS for 15 days. Fresh supernatant was added after each recording to perform cumulative release over time (n = 3 error bars correspond to standard deviations).

FIGURE 5

Rheological properties and stability of both gelatin (Gel) and Gelatin NPs composite (Gel-PDA-PAR₃₀) hydrogels. (A) Shear viscosity as a function of shear rate for both Gel and Gel-PDA-PAR₃₀. (B) Storage modulus (G’) as a function of frequency for both conditions tested (Gel vs. Gel-PDA-PAR₃₀). Stability of the different hydrogels tested (Gel vs. Gel-PDA-PAR₃₀) at 37°C in a relevant biological medium, (C) enzymatic stability in collagenase; (D) in acidic condition (pH = 5) and in (E) physiological pH (pH = 7.4). The results are expressed as a percentage of remaining mass at different time points (n = 3 error bars correspond to standard deviations).

FIGURE 6

(A) Normalized S. aureus growth in supernatant after 24 h in contact with Gelatin NPs composite hydrogel loaded with NPs (Gel-PDA-PAR₃₀) at different dilutions (n = 3 and error bars correspond to standard deviations) (***p < 0.001). (B) SEM images of Gelatin vs. Gel-PDA-PAR₃₀ 1:4 (NPs diluted 4 times in the hydrogel matrix) after 24 h of exposition with S. aureus.

FIGURE 7

Cytotoxicity test with Balb 3T3 cells of Gelatin NPs composite hydrogel loaded with NPs (Gel-PDAPAR₃₀) at different dilutions to determine the threshold of NPs toxicity while keeping the antimicrobial activity of the resulting hydrogel (n = 3 and error bars correspond to standard deviations).