# Transparent, Pliable, Antimicrobial Hydrogels for Ocular Wound Dressings
## 3 Results
### Carboxylated Agarose Allows Control Over Release Profile of Antimicrobial Agents  
The cumulative release profiles of cefazolin and doxycycline from hydrogel samples into a PBS solution was measured over the course of 12 h (Figure 5A,B). For both antimicrobials, the introduction  
Figure 4: (**A**) Plot of Young’s moduli versus wt.% of CA in total weight of polysaccharide. A near-linear relationship can be observed. (**B**) Young’s moduli of hydrogel blends loaded with cefazolin and doxycycline, respectively, compared with antimicrobial-free blends (blank hydrogels). Suggesting that a 0.5% load of antimicrobial agents does not affect the modulus of an NA–CA hydrogel. (**C**) Flexibility of the hydrogel pads assessed on a human eye. Blue arrow shows the increased curvature of the hydrogel as the concentration of CA increases. of CA increased the release rate. The addition of 50% of CA in the formulation NA50 CA50, led to an 81% total release of cefazolin within the first 6 h. The release after 12 h was complete only in the hydrogels formulated with CA. For the doxycycline, the release was slower and after 12 h only 80% of the drug was released in the NA50 CA50 formulation. Nevertheless, the effect of CA on acceleration of diffusion of antimicrobial molecules from hydrogel samples into PBS is here clearly demonstrated. While the two antimicrobials are water soluble, at pH 7.4, most carboxylic acid groups on cefazolin will be deprotonated resulting in an overall negative charge. Conversely, doxycycline will be in the zwitterion and might interact with the negative charges of the CA [28]. When compared with doxycycline, the negative charge on cefazolin could explain the faster release from the negatively charged CA network. Electrostatic repulsions between the charged polysaccharide network and the charged molecule could influence the release rate. For the application of these hydrogel blends as a wound dressing, a continuous antimicrobial activity during the application window is desirable. As a wound dressing, the hydrogel devices form a barrier to prevent infection. Additionally, hydrogels due to their aqueous environment are an ideal environment for bacterial growth. Supplementing the CA/NA blends with antimicrobial agents offers the possibility to avoid bacteria growth in the hydrogel but also around the hydrogel as the antimicrobial agent diffuses outside. First, the minimum inhibitory concentration (MIC) for the antimicrobial molecules was determined against Gram negative (_E. coli_) and Gram positive (_S. aureus_) strains. The MICs for cefazolin and doxycycline against _E. coli_ were 2 ug/mL and 0.25 ug/mL, respectively. Similarly, for _S. aureus_ the MIC was 0.5 ug/mL for cefazolin and 0.25 ug/mL for doxycycline. To characterize the efficacy of the blend formulations we performed a zone of inhibition (ZOI) assay. Each of the antimicrobials was loaded in the different hydrogel formulations and the potency of the different formulation was evaluated. As expected from the MIC and release kinetics, cefazolin and doxycycline lead to a broad ZOI and both antimicrobials are more efficient against _S. aureus_ than _E. coli_. While in the release experiment, we observed slight differences in the release rates of the antimicrobials across the different formulations, on both strains we obtained a similar ZOI across the three formulations. All three formulations resulted in similar ZOI against both strains. The ZOI  
Figure 5: Release profiles of (**A**) cefazolin and (**B**) doxycycline in phosphate-buffered saline (PBS). The effect of CA on the acceleration of the release rate is apparent for the two types of antimicrobial agents tested. (**C**) Zone of inhibition of the three hydrogel formulations each loaded with 3 mg/mL of antimicrobial and tested on _Escherichia coli_ and _Staphylococcus aureus_. (N = 3, standard deviation). against _E. coli_ ranged between 28.2 and 30.2 mm for cefazolin and between 26.5 and 27.6 mm for doxycycline. ZOIs against _S.