As previously mentioned on the methodology section, our patch consists of a multilayer matrix composed of: (i) Poly(vinyl alcohol), which serves as a protection mechanism of the complete system and also allows in/out oxygen transfer. (ii) petrolatum, which is a chemical that prevents the polymer to be directly in contact with the culture medium and also avoids the matrix to be dissolved. (iii) Nutritive Agar + PVA, the former provides nutrients and medium for the microorganism, while the latter is added as a way to preserve the medium. (iv)Nitrocellulose membrane (0.22 μm) allows the diffusion of bacteriocin and alginate lyase, but avoids the spreading of the bacteria towards the injury. (v) Autoadherible polymer, which holds the system hermetically. Poly(vinyl alcohol) (PVA) is a water-soluble synthetic polymer produced via hydrolysis of polyvinyl acetate to remove acetate groups. PVA film is a biomaterial that shows properties like inertness and stability, and it is considered safe and biocompatible in medicine and pharmacy (Muppalaneni and Omidian, 2013). In order to obtain the PVA film with suitable characteristics for our application; two experiments of polymerization were performed. In experiment 1, the polymer was dissolved in water; while in the experiment 2, HCl was used as a catalyst for the reaction of polymerization. In experiment 1 (no addition of HCl), all samples were able to form an elastic film, when drops of water were added the film tended to soften and with an excessive amount of water, the film became softener. In experiment 2 (wherein HCl was used as a catalyst), sample with only 100 μL of HCl appeared to be harder than samples from experiment 1 but it displayed more air bubbles within its structure. With 200 μL of HCl, the film showed less softening compared to those from the experiment 1. Even though the addition of HCl conferred waterproofness to the film, air bubbles were a major issue, so it was decided that the use of this acid was unnecessary. Therefore, for future assays, the mixture was heated at the conditions of time and temperature of the experiment 1 (60°C for 40 minutes), and afterwards it was poured all over an aluminium foil and kept at room temperature for 24 hours. With this procedure a firm and promising film was obtained to be used in the biopatch.
Selection of agar medium concentration
The culture medium is an essential part for the product that we are trying to develop because it is going to provide the nutrients demanded by the microorganism; furthermore it is a matrix where our sensor and machinery will be placed. We observed cell motility of E. coli and appearance of agar concentrations of 2.5, 3, 3.5, 4 and 4.5 g/L. All of them presented good cell motility but the one that seemed to be more consistent was 3.5 g/L; 4 and 4.5 g/L had a solid consistency instead of a semisolid (data not shown) and lower concentrations looked more liquid (table 1).
Table 1. Motility on semisolid medium at 2.5, 3 and 3.5 (g/L) agar concentration.
Selection of PVA medium concentration
For greater preservation of the culture medium which will contain the genetically modified bacteria, we used a combination of culture medium (optimal agar concentration of 3.5 g/L obtained in this work) and various concentrations of the polymer PVA (5, 10 and 15%, v/v), from the PVA obtained under the conditions determined in this work as ideal. The preservation of the culture medium was measured based on the height that it presented. According to the medium heights obtained, it was decided not to use polymer in the medium due to the low decrease of height in the negative control after ten days compared to those where certain amounts of PVA were added. Also, we observed that in the last day of the experiment (12th day) the heights of the medium with 5% of PVA and the negative control (no PVA added) were similar (Figure 6).
Figure 6. Decrease of media heights over time in the presence of different PVA concentrations.Negative control had no PVA at all.
Membrane permeability for microorganisms
In order to prove that microorganisms are not able to pass through the nitrocellulose membrane which has a pore diameter of 0.22 µm, we simulated the biopatch being in contact with skin. To do so we inoculated nutritive agar medium with E. coli and then covered it with the membrane and two filter paper pieces in order to simulate skin. From experiment 1, samples did not show growth both in the tubes containing the filter paper pieces (Figure 7a) and in the Petri dishes where filter paper 1 was put. The only exception was sample 3 (Table 2) which came out to be contaminated due possibly to a deficient manipulation technique during the procedure, even in the initial Petri dish more colonies than inoculated appeared. From experiment 2, exactly as for experiment 1, all samples did not show bacterial growth with the exception of one, S1, which also displayed more colonies than inoculated at the beginning (Table 2). As expected, for the positive controls, where no membrane was added, tubes containing its filter paper pieces (1 and 2) revealed bacterial growth (turbid broth) as well as the Petri dish where filter paper 1 was put. All negative controls had no growth at all since no microorganism was inoculated (Figure 7b).
These experiments validate that the nitrocellulose membrane with a pore diameter of 0.22 μm does not allow bacteria to move from the biopatch towards the burn injury because the size of E. coli is approximately 1 to 3 micrometers (Reshes et al., 2008).
Figure 7. Evaluation of membrane effectiveness to avoid flow of microorganisms. A) Sample 1 from experiment 1, filter paper in nutritive broth after 48 hrs, no turbidity, negative growth). B) Negative Control 1 from experiment 2, filter paper in nutritive broth after 48 hrs, no turbidity, negative growth.
Table 2. Results of the experiment of membrane permeability for E. coli.
PVA films were developed by a simple dissolution in water or in aqueous medium adding HCl to catalyze the reaction. Films were formed through all the tested conditions and showed a transparent appearance and elasticity in certain degrees; however, in those where HCl was used, different quantities of bubbles were formed and this condition could affect in a negative way film mechanical properties. From the aforementioned, we consider the best films were those performed through the aqueous treatment without HCl. On the other side, in order to develop a culture medium which was able to provide nutrients and bacterial motility, different agar concentrations were tested, resulting the 3.5 g/mL concentration that with the most solid consistence, which still permitting bacteria motility. Furthermore, hydrogels containing different PVA/Agar proportions were prepared, and it was found those having only agar showed less reduction in height, from this fact we inferred these ones could maintain for a longer time to bacteria in stable conditions. Additionally, 0.22 µm-membrane effectivity (which was included in patch design) was evaluated to verify if this really avoids microorganism diffusion and it was found that it is functional. This assay was made in order to determine if the aforementioned membrane will exclude microorganisms flow from the patch to the burns and vice versa.