*3.8. In Vitro Biological Properties*

### 3.8.1. Antimicrobial Studies

Antibacterial hydrogel wound dressings have been developed with the consideration of the fact that moist, warm, and nutritious environments in wound beds provide ideal conditions for microbial growth.

The hydrogels based on CS/PVA/TA presented significant antibacterial properties against *E. coli* and *S. aureus* with an inhibition rate between 95 and 97% [70]. The antimicrobial activity of SSD-alginate hydrogel and SSD-alginate-PG hydrogel produced using γ-irradiation at 2.5 kGy was determined against most encountered bacterial strains in burn wound infection such as *E. coli*, *K. pneumoniae*, *P. aeruginosa*, *A. baumannii*, *E. aerogenes*, and *S. aureus*. The irradiation treatment improved the performance of the formulated hydrogels and the highest inhibiting bacterial growth was observed for SSD-hydrogel loaded with prodigiosin known as an active ingredient with important antifungal, antibacterial, antimalarial, and antineoplastic properties [109]. CPC/PVA hydrogels demonstrated antibacterial activity on the *E. coli* strain, but this property is due to the action of cetylpyridinium chloride [96]. Regarding the antimicrobial effects of gelatin/PVA hydrogel in which various concentrations of AgNP were added, it was demonstrated that a proper concen-

tration of 0.25% AgNP is efficient against *E. coli* and *S. aureus*, as well as MRSA. These values are considered a balance between biological and mechanical properties [63]. The CIP/PLGA-loaded PVA hydrogels denote important antibacterial activity in the *E. coli* and *S. aureus* strains [67]. The hydrogels fabricated from PVA and ws-chitosan showed efficiency only on *E. coli* due to their capability to bind Gram-negative bacteria to the chitosan amino groups [100]. PVP/carrageenan hydrogels incorporated with nanosilver prepared at 25 kGy by in situ reduction of Ag<sup>+</sup> and hydrogel crosslinking using γ-irradiation showed antimicrobial activity against the common contaminants isolated from burn patients, *P. aeruginosa*, *S. aureus*, *E. coli*, and *C. albicans*. The most effective hydrogels against bacterial strains were those loaded with 100 ppm nanosilver after 3–6 h [102]. PVA/PVP/glycerin/antibacterial hydrogels produced by irradiation at 25 kGy and freeze-thawing were verified for their efficiency against the three bacterial strains *E. coli*, *S. aureus*, and *S. aeruginosa*. In this case, the antibacterial effect generally decreased after irradiation. The hydrogels containing chloramine-T showed an accentuated decrease of antibacterial activity due to its weak resistance at irradiation. Upon irradiation can be generated radiolysis product, which reduces its bactericidal effect. It must be specified that the antibacterial properties of hydrogels containing sulfadiazine sodium salt were not affected by the γ-irradiation [98]. Quaternary ammonium chitosan hydrogel blended with PVA and PEO performed great antibacterial activity against *S. aureus* and *E. coli* [76]. The quaternary ammonium chitosan exhibits important antibacterial activity due to its polycationic structure which is attracted to the surface of bacteria and leads to the alteration of their cell membrane, releasing a significant amount of protein material or cytoplasmatic constituents out from the bacterial cell, finally producing the death of the bacteria [149]. Xanthan-based wound dressing hydrogels containing ZnO nanoparticles crosslinked at 30 kGy were tested to demonstrate the antibacterial activity against *S. aureus*, *E. coli*, and *C. albicans*. The results indicated a broad antimicrobial spectrum, both antifungal and antibacterial, and were correlated with the increase of ZnO nanoparticles included within xanthan dressings [114]. The nanosilver/gelatin/CM-chitosan hydrogels clearly showed antibacterial ability against *E. coli.* The zone of inhibition was wider at 10 mM of nanosilver [62]. The radiation synthesized of 2-(dimethylamino)ethyl methacrylate/PEO/ZnS nanocomposite hydrogel at 20 kGy loaded with antibiotics (colistin, gentamicin, and neomycin) was tested against both bacterial strains (*E. coli*, *P. aeruginosa*, *S. aureus*, and *B. subtilis*) and unicellular fungi (*A. niger*, *A. terreus*, *A. flavus*, and *A. fumigatus*). The presence of ZnS nanoparticles indicates the increase of antimicrobial and antifungal activity on all strains tested, especially those hydrogels loaded with neomycin [71]. PVP/chitosan/itaconic acid/ZnO nanocomposite hydrogel was efficient against *S. aureus* and *E. coli*. The incorporation of amoxicillin into these hydrogels increased significantly the antimicrobial activity [91]. Strong antimicrobial effect and complete inhibition of *P. aeruginosa*, *S. aureus*, and *C. albicans* were observed in the case of PVP/alginate hydrogel dressings containing 70 ppm nanosilver crosslinked by γ-irradiation at 25 kGy [38]. Smart P(HEMA/IA/silver NP) hydrogels were produced in the same step with the reduction of Ag+ by the γ-irradiation method at 12 kGy. The Ag/P(HEMA/IA) hydrogel demonstrated high antibacterial effects on *E. coli*, *S. aureus*, and *C. albicans* with effectiveness even at small silver concentrations [64]. AA/ZnCl<sup>2</sup> crosslinked hydrogels at 25 kGy indicated that they have good antimicrobial activity against *S. aureus* and *E. coli* strains. The antibacterial effects of AA/ZnCl<sup>2</sup> hydrogels increased as a function of the ZnCl<sup>2</sup> concentration [79]. Antimicrobial hydrogel wound dressing was developed, where they included CS dissolved in lactic acid to the original composition of PVP/Agar hydrogel dressing before irradiation. The Gram-negative bacteria *E. coli* growth was not inhibited and the test involving *S. aureus* showed that Gram-positive bacteria growth was hindered in the presence of hydrogel dressings containing CS as compared with the original hydrogel dressing [120]. The synthesized PVP/AgNPs hydrogel showed antimicrobial activity against *P. aeruginosa* and *S. aureus* and the PVA/AgNPs hydrogel indicated that it only had antibacterial activity against *P. aeruginosa* [83]. In another work, the pure PVP hydrogels had no inhibition zone, while the PVP/AgNPs hydrogels (5 mM) presented

inhibition zones against all tested bacteria: *B. cereus*, *S. aureus*, *S. epidermidis*, *S. pyogenes*, *A. iwoffii*, *E. coli*, and *P. aeruginosa*. The PVP/AgNPs hydrogels with different concentrations of AgNPs irradiated at 25 kGy were investigated for bactericidal efficacy against *S. aureus*, it was proven that the rate of bacterial reduction increased with the increase of AgNPs content up to 99%, over 6 h (5 mM AgNPs) and 12 h (1 mM AgNPs) [65]. The Psyllium/Carbopol hydrogel dressings with frankincense essential oil developed using γ-irradiation showed efficient antimicrobial activities against *S. aureus*, *E. coli*, and *C. albicans* [150].

The antibacterial activity data for different hydrogel wound dressings are presented in Table 4. The in vitro antimicrobial tests of hydrogels were explored using microorganisms on Gram-positive bacteria (*S. aureus*, *C. albicans*) and Gram-negative bacteria (*P. aeruginosa*, *E. coli*).


**Table 4.** Antibacterial activity data of the hydrogel wound dressings.

### 3.8.2. Cytotoxicity Studies

It is important to underline that from our study it emerged that the most used polymer for the development of hydrogels by radiation crosslinking for dressing applications is poly(vinyl alcohol). Cytotoxicity studies showed γ-crosslinked PVA hydrogel exhibited partial toxic effects on the L929 fibroblasts by comparison with PVA hydrogels crosslinked with glutaric aldehyde. This behavior has been associated with the partial crosslinking of the PVA polymer and some degradation products resulting after the irradiation process, or it is due to the high radiation dose used for the crosslinking reaction [152].

Considering that wound dressing hydrogels generally have as their main application the healing of skin wounds, this review takes into account the works that refer to the testing of these materials from the point of view of their cytotoxicity on skin-specific cells. The in vitro biological properties of BC/AA hydrogels were assessed by cytotoxicity test on human fibroblast cells. After a 24 h incubation, the hydrogels formulation was found nontoxic and showed cell viability above 88% [50]. The extract of PVA/ws-chitosan/glycerol hydrogels was nontoxic towards L929 mouse fibroblasts; moreover, they showed an accelerated wound healing process [89]. The cytotoxicity test using the NCTC Clone 929 cell line on the PVP hydrogels reinforced with PP showed cell viability above 75%. The percent of cell

viability depended on the extract concentration [56]. PVA/alginate hydrogels, after incubation for 48 h, showed a considerable reduction in toxicity on L929 fibroblasts. All hydrogels tested showed a cell viability greater than 85% [84]. The cytotoxicity of AgNP/Gelatin/PVA hydrogels was investigated on normal human dermal fibroblasts showing 80% cell viability. The cell viability was little influenced by the increase in AgNP concentration and the incubation period [63]. The toxicity of CIP/PLGA-loaded PVA hydrogel prepared by γ-irradiation at 25 kGy was tested on human dermal fibroblasts. The studies indicated the non-toxicity of 244 mg CIP/PLGA-loaded PVA hydrogels [67]. In vitro cytotoxicity studies of gelatin/poly (γ-glutamic acid) hydrogels performed on the L929 fibroblasts revealed no cytotoxicity [74]. PVA/ws-chitosan/glycerol hydrogels produced by combining γ-irradiation at 40 kGy with a freeze-thawing cycle demonstrated non-toxicity towards L929 mouse fibroblasts. Moreover, these hydrogels showed cell viability higher than 100% after 24 h of incubation [101]. The ELT/PVA hydrogel demonstrated high cell viability (99.4%) on calcein and ethidium homodimer fibroblasts at lower concentrations (0.10% *w*/*v* ELT), whereas higher concentrations of ELT (0.15–0.25% *w*/*v*) revealed low values of viability [72]. The CM-CS/PEDGA hydrogels did not show significant cytotoxicity on the mouse fibroblast cell line L929, and even an increase in the number of cells was observed at lower extract concentrations (84.3%) [153]. PVA/sericin hydrogels were evaluated to determine the cytotoxicity of an indirect method on L929 mouse fibroblast cells. The cells cultured with the extracts of PVA/sericin hydrogels containing a higher sericin content (25% *w*/*v*) had proliferated more than cells cultured with the extracts of hydrogels containing a lower sericin content [147]. The PVP/AgNPs presented a low toxicity to human fibroblast cell line HaCaT of approx. 10%. For the PVA/AgNPs, cellular death was observed in up to just over 20% of the tested fibroblasts, so the hydrogel could be considered cytotoxic. Therefore, the PVA and PVP hydrogels with AgNPs were shown to be biocompatible, and they were considered nontoxic [83]. The effectiveness of the PVP/AgNPs hydrogels as a wound dressing was investigated for their cytotoxicity using mouse fibroblast cells (L929) and these hydrogels presented cell viability greater than 80% [65]. Cell viabilities for PVA/GEL bilayer hydrogel using 3T3-Swiss albino mouse embryonic fibroblasts were 98% (GEL) and 88% (PVA), respectively, thus showing good biocompatibility [138].

### **4. General Remarks and Future Perspectives**

The scientific literature presents hydrogel wound dressings as promising candidates for improving traditional methods in various biomedical applications. Different advanced concepts have been developed in the last twenty years to prepare unique and biocompatible hydrogel wound dressings mainly using ionizing radiation.

This review paper includes methods of characterizing polymer hydrogels crosslinked with ionizing radiation. Furthermore, the paper includes general results published by different research groups that have studied the behavior of hydrogels used as wound dressings.

Each characterization method is essential and helps to obtain a hydrogel with properties that mimic human skin and its healing/regeneration capacity as faithfully as possible. In addition to crosslinking, the irradiation technology ensures dressing sterilization, a fundamental requirement for medical devices that come into contact with skin wounds. Additionally, this review paper includes studies on the antimicrobial and cytotoxicity properties of various hydrogels. Cytotoxicity studies represent an efficient method of testing cell proliferation and are discussed by several authors.

This review paper demonstrates that the hydrogels developed by radiation technologies are excellent dressing materials for wound healing. The crosslinked hydrogels in this way can absorb wound fluids, have good antimicrobial properties, improved functional adhesion, are cost-effective, and are easy to remove.

Hence, the polymers, antibacterial agents, drugs, and radiation synthesis used significantly improved the physicochemical properties of the prepared hydrogel dressings. Since e-beam and γ-irradiation are common sterilization methods, the properties of the irradiated hydrogels at doses of 25–30 kGy were investigated.

In the specialized scientific literature, a small number of clinical studies used hydrogel wound dressings. Therefore, it is necessary to carry out new such studies considering the useful properties of hydrogel dressings obtained by irradiation. The future development of hydrogel wound dressings will lead to much more precise and intelligent properties, biocompatibility, and well-regulated antibacterial activity.

**Author Contributions:** Conceptualization, M.D., I.C. and A.S.; data curation, M.D., I.C. and A.S.; writing—original draft preparation, M.D., I.C. and A.S.; writing—review and editing, M.D., I.C. and A.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by a grant from the Ministry of Research, Innovation and Digitization, CNCS—UEFISCDI, project number PN-III-P1-1.1-PD-2021-0552, within PNCDI III, as well as from the Romanian National Core Program LAPLAS VII–contract no. 30N/2023.

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

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** No new data were created or analyzed in this study. Data sharing is not applicable to this article.

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

### **Abbreviations**



### **References**


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