*3.3. Swelling and Contact Angle*

Figure 5A shows the swelling curves in water at 25 ◦C; PVC has a hydrophobic character for what it did not swell. Samples modified only with 4VP showed their maximum swell at 2 h. On the other hand, the catheters with 4VP/4VPPS reached their maximum swelling at 30 min and presented maximum percentages three times those reached by the samples without functionalization. Figure 5B shows the contact angles for the different materials; the PVC surface showed a contact angle of approximately 100◦, which corresponds to its hydrophobic character; the modified materials, on the other hand, presented contact angles of less than 90◦, indicating that the surface acquired hydrophilicity. The surfaces modified with 4VP/4VPPS initially had higher contact angles than those modified only with 4VP; however, after 10 min of water-surface interaction, the contact angles decreased to similar values.

**Figure 5.** (**A**) Swelling curves in water at 25 ◦C, reported: mean ± standard error of the mean, *n* = 3. and (**B**) Water contact angle, reported: mean ± standard error of the mean, *n* = 6. PVC-*g*-4VP4%/4VPSS32% (4/32), PVC-*g*-4VP10%/4VPSS13% (10/13), and PVC-*g*-4VP16%/4VPSS22% (16/22).

### *3.4. pH-Sensitivity*

4VP is a pH-sensitive polymer because it has an amino group in its structure that presents an acid-base balance (Figure 6A), with a pKa around 5.4. At pHs below its pKa, the polymer is in its ionic form, so the chains suffer repulsion between them and swelling is greater. Figure 6B shows the behavior of the swelling as a function of the pH for the pristine PVC, PVC-*g*-4VP, and PVC-*g*-4VP/4VPPS. PVC did not present a pH response, whereas all the materials modified with 4VP showed this capability, with a critical pH in the range of 6.3 to 7.

**Figure 6.** (**A**) Acid-base balance of 4VP and (**B**) pH-sensitivity, reported: mean ± standard error of the mean, *n* = 3.

### *3.5. Protein Adsortion Test*

The presence of the 4VPPS zwitterionic polymer decreased the percentage of protein adsorbed on the surface. Figure 7 shows the results; the material modified with 10% 4VP and 13% 4VPPS was the one that showed the highest antifouling capacity, reducing BSA adsorption by 74% compared to the unmodified PVC surface. This shows that the functionalization of the grafted 4VP was successful and the 4VPPS formed expresses its antifouling characteristics.

**Figure 7.** Protein adsorption on the polymer surface of different composition (incubation: 30% BSA solution in PBS, 37 ◦C, 2 h). Reported: mean ± standard error of the mean, *n* = 3.

### *3.6. Load and Release of Ciprofloxacin*

Ciprofloxacin loading and release assays were performed on PVC, PVC-*g*-4VP(4%), PVC-*g*-4VP4%/4VPPS32%, PVC-*g*-4VP10%/4VPPS13%, and PVC-*g*-4VP16%/4VPSS22% samples. PVC and PVC-*g*-4VP(4%) samples did not show ciprofloxacin loading capacity, contrary to the samples modified with the zwitterionic polymer. Figure 8A shows the ciprofloxacin load for the three zwitterionic graft samples; in all the cases, the load reaches maximum levels after 30 h of interaction, but the amount of ciprofloxacin loaded by the PVC-*g*-4VP10%/4VPSS13% and PVC-*g*-4VP16%/4VPSS22% catheters was higher. Figure S4 shows the load profiles. However, release profiles (Figure 8B), showed that the sample PVC-*g*-4VP10%/4VPPS13% had a final release of approximately 40 μg/mL, and the samples PVC-*g*-4VP4%/4VPPS32% and PVC-*g*-4VP16%/4VPPS22% released around 20 μg/mL; in all cases, the release was gradual until 30 h.

The release profiles were processed using DDSolver Excel software to determine the fitting model (details of the fit are specified in Supplementary Materials, Table S2). In all cases, following the values of the Akaike Information Criterion (AIC), the Model Selection Criterion (MSC), and the adjustment of squares (r2), the model that presented the highest affinity with the behavior of the materials was the Peppas–Sahlin model [39].

**Figure 8.** (**A**) Ciprofloxacin load (condition: 0.012 μg/mL, 25 ◦C por 30 h) and (**B**) Ciprofloxacin release profiles (condition: PBS a 37 ◦C). Reported: mean ± standard error of the mean, *n* = 3.

The Peppas–Sahlin model describes systems that present the contribution of two mechanisms (diffusional and relaxational) in the release process. This model is described by Equation (4), where M∞ is the amount of drug in an equilibrium state, Mt is the amount of drug released in the determined time, k1 represents the Fickian diffusion contribution, k2 the polymer chains relaxation contribution, and m is the Fickian diffusion exponent [40].

$$\mathbf{M}\_{\rm l}/\mathbf{M}\_{\infty} = \mathbf{k}\_1 \mathbf{t}^{\rm m} + \mathbf{k}\_2 \mathbf{t}^{\rm 2m} \tag{4}$$

Table 3 shows the values of the Peppas–Sahlin model parameters for the different materials; all materials presented values of k1 that predominate over k2, indicating that the principal release mechanism is diffusion. However, for the material, the contribution of the chain relaxation mechanism is higher, possibly due to the greater amount of the zwitterionic polymer. The PVC-*g*-4VP10%/4VPPS13% catheter released the highest amount of ciprofloxacin, with a release rate of approximately 60%.

**Table 3.** Peppas–Sahlin model parameters.


The PVC-*g*-4VP10%/4VPPS13% materials presented the best properties and fast synthesis conditions (0.35 M, 70 ◦C, 5 min, and 12% 4VP), which is why they were chosen to carry out cell viability, antimicrobial activity, and antifouling capacity tests.

### *3.7. Cell Viability*

Figure 9 shows the results of cell viability using BALB/3T3 murine embryonic fibroblasts; it was observed that the control PVC increased cell growth, in the same way as the material modified with 4% 4VP. The material grafted with 4VP/4VPPS showed a decrease in cell growth of approximately 5%; when the material was loaded with ciprofloxacin, the cell growth decreased by 18%; however, the material can be considered non-cytotoxic according to ISO-10993-5-2009 [41].

**Figure 9.** Cell viability result using BALB/3T3 murine embryonic fibroblasts and MIT assay. Reported: mean ± standard error of the mean, *n* = 3.

### *3.8. Antimicrobial Activity and Antifouling Capability*

Figure 10A presents the antibacterial capacity of the material based on its ability to inhibit the growth of *E. coli*. It was observed that PVC, PVC-*g*-4VP(4%), and PVC-*g*-4VP10%/4VPPS13% do not have antimicrobial activity, on the contrary, PVC-*g*-4VP10%/ 4VPPS13% loaded with ciprofloxacin achieved 82% inhibition at 24 h. On the other hand, Figure 10B presents the antifouling capacity of the material determined by the number of colony-forming units of *E. coli* bacteria, which adhered to the material when it was in direct contact with the *E. coli* solution of 1 × <sup>10</sup><sup>8</sup> cfu for 24 h. It is observed that the material with binary graft presents a 42% decrease in bacterial adhesion in the control PVC and this effect is enhanced when loading ciprofloxacin, reaching a 55% decrease.

**Figure 10.** (**A**) Inhibition of bacterial growth for *E. coli* and (**B**) Bacterial adhesion of *E. coli.* Reported: mean ± standard error of the mean, *n* = 3.

### **4. Discussion**

Ionic polymer functionalization using oppositely charged ions is one of the ways to synthesize zwitterionic polymers. In this case, the functionalization of 4VP grafted onto PVC catheters made it possible to obtain the characteristics of the 4VPPS zwitterion while maintaining the pH-sensitivity of the 4VP system. The 4VP is a cationic polymer that reacts with PS to produce 4VPPS, by a ring-opening alkylation reaction (Figure 1). This form of zwitterion synthesis resulted in different percentages of grafting, which was impossible with a direct grafting method, that is, directly grafting the 4VPPS monomer. The difficulty of working with the 4VPPS monomer was due to its insolubility in most solvents; a 1 M saline solution is required to solubilize it [42,43]. The formation of grafts using ionizing radiation represents an advantageous method for medical device modification because the obtained materials do not present contamination by agents used for the polymerization [44,45].

The poly (4VPPS) is an aromatic zwitterionic polymer that has demonstrated antifouling capabilities in biomedicine, electronics [46], and water treatment. Venault, et. al. synthesized 4VPPS hydrogels to be used as medical device coatings. They demonstrated that 4VPPS coatings present a bio-antifouling capability similar to that of sulfobetaine methacrylate (SBMA), one of the most common zwitterionic polymers, employed to produce antifouling surfaces by coating, grafting, or copolymerization [47–50]. Additionally, 4VPPS coatings were more resistant to temperature than SBMA coatings because they retained their bio-antifouling properties after being heated at 121 ◦C for 1 h (sterilization conditions), representing an advantage in the biomedical field, where most of the devices require sterilization [25]. On the other hand, Gui, et. al. copolymerized acrylamide with 4VPPS to produce a zwitterionic poly-acrylamide that showed thermic stability and good properties as a flocculant of cationic and anionic inorganic contaminants due to its electrostatic characteristics [51].

Materials grafting with 4VPPS showed the ability to load and release ciprofloxacin due to the hydrophilicity and pH-sensitivity of the system. In addition, the modification provided the material with the antifouling property of zwitterionic polymers, allowing a significant decrease in both the adhesion of the BSA protein and *E. coli*. A synergistic effect was observed when loading the material with ciprofloxacin, increasing its capacity to prevent the adhesion of *E. coli* by 13%, for the PVC-*g*-4VP10%/4VPPS13% catheter. The materials were also shown to be non-toxic since they did not significantly affect cell viability. The modification of the catheters allows the creation of antimicrobial systems with the capacity to release an antibiotic at the site of interest, making it a localized drug delivery system, increasing its effectiveness and decreasing the side effects.

### **5. Conclusions**

The functionalization of the 4VP grafted onto PVC catheters was successful and obtained materials with different percentages of 4VP and 4VPPS in their composition. Although all the modified catheters showed hydrophilicity and pH sensitivity, the PVC-*g*-4VP10%/4VPPS13% catheter was the one that showed the best capacities for loading and releasing ciprofloxacin, achieving an inhibition of *E. coli* growth of approximately 82% in 24 h. In addition, due to the presence of zwitterion, this material showed antifouling characteristics, which decreased the adhesion of BSA by 74% and the adhesion of *E. coli* by 55%. This material is a pH-sensitive system with dual antimicrobial capacity and is therefore a promising alternative for developing devices with less tendency towards biocontamination.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/pharmaceutics15030960/s1, Figure S1: Calibration curves to quantify load of ciprofloxacin; Figure S2. Calibration curves to quantify release of ciprofloxacin; Figure S3. Calibration curve to BSA quantification (Abs 556 nm); Figure S4. Profiles of ciprofloxacin load; Table S1: Working solution preparation; Table S2. Parameters to select the drug release model.

**Author Contributions:** Conceptualization, L.D-P. and E.B.; methodology, L.D.-P. and H.M.; formal analysis, L.D.-P.; investigation, L.D.-P.; resources, E.B. and H.M.; writing—original draft preparation, L.D.-P.; writing—review and editing, E.B. and H.M.; visualization, L.D.-P.; supervision, E.B.; project administration, E.B.; and funding acquisition, E.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the Dirección General de Asuntos del Personal Académico (DGA-PA), Universidad Nacional Autónoma de México under Grant IN204223.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** L. Duarte Peña (887494) acknowledges CONACyT for the doctoral scholarship. This work was supported by DGAPA-UNAM [Grant IN202320] (Mexico). Benjamin Leal and Martín Cruz from ICN-UNAM are acknowledged for their technical assistance.

**Conflicts of Interest:** The authors declare no conflict of interest.
