Green-synthesis of copper oxide nanoparticles (CuO NPs)

The greenly synthesized CuO NPs were successfully prepared and fully characterized in our previous published work [21]. SEM results showed spherically shaped particles with around 230 nm in diameter.

Film formation and optimization

Simple visual inspection of plain AFs and AZFs prepared with/without CaCl2 treatment (control films) indicates that all films are flexible, transparent, homogenous, with no evidence of air bubbles or agglomerations and pliable (Fig. 1A). While the yellow shade of loaded films comes mainly from the intrinsic color of CuO powder (Fig. 1A). In the preparation of the ‘blinding films’, CaCl2 solution was mixed directly into alginate solution and then casted into films.

Fig. 1

(A) Photographed images, (B) FTIR patterns and (C) SEM images of blank AF (F5), blank AZF (F6), 0.4% w/v CuO NPs-loaded AF (F7), 0.1% w/v CuO NPs-loaded AZF (F8), 0.2% w/v CuO NPs-loaded AZF (F9), and 0.4% w/v CuO NPs-loaded AZF (F10)

In the second dipping method, blinding films were then soaked in 5%, w/v CaCl2 solution for 5 min. After soaking, the films were found to dry much faster than the control or blending films, resulting in slightly translucent, stiff films, referred to as “dipping films”. The quality of the control and blinding films was not good, and they can readily degrade totally in an aqueous medium within 5 min. This may be credited to the hydrophilic nature of alginate, as well as possibly to the inadequate gelation with a small amount of Ca2+ elicited partial crosslinking and faster hydrolysis. Conversely, better film quality was attained from the dipping films; this may be appertaining to the crosslinking of alginate molecules with sufficient (Ca2+) molecules, producing water-insoluble crosslinked calcium alginate films by the ion exchange reaction which, in turn, facilitated their subsequent fabrication into dimensionally firm films. Dipping treatment enriched the mechanical integrity of the “blinding” films [6, 34]. Dipping films preparation method was chosen as the optimized formulation for further CuO NPs loading and evaluation.

Characterization of the tailored filmsFilm thickness

The film thickness was significantly dependent on the film composition and their preparation method (Table 2). It was observed that the thickness of the ‘blending films’ is recorded of (179–196 mm), followed by the control (169–189 mm) and then the ‘dipping films” (168–188 mm). The thickness of the dipping films seems to decrease as some alginate molecules were crosslinked by Ca2+ which occurred at the surface of the films, resulting in more packed molecules with a reduction in thickness [6]. However, the incorporation of zein into AFs interprets a significant (P < 0.05) thickness increase in all methods (F2, F4, F6); compared to the plain AFs. This behavior may be associated with the differences in the molecular masses of the biopolymers. It was previously reported that zein films were thicker than alginate films constituting the same concentrations and volumes [35]. The thickness of the films did not significantly change with the presence of CuO NPs (P > 0.050).

Table 2 Study of water permability, swelling ratio (%) and degradation (%) of films coded (F1-F10)FT-IR analysis

The spectrum of blank alginate film (F5) shows two peaks at ν 3334, and 2925 cm− 1 assigned to stretching vibrations of OH, and CH bonds: respectively. The peaks at ν 1086 and 1027 cm− 1 are attributed to the stretching vibration of C-O-C bond (Fig. 1B). The asymmetric COO− vibrational peak shifted from ν 1595 to 1605 cm− 1, while the symmetric COO− peak shifted to a higher wavenumber (from ν 1408 to 1422 cm− 1). This can be accredited to the interaction between carboxylic group and calcium ions, because of the crosslinking [34, 36].

Upon mixing zein with alginate, (F6), a peak band at ν 2960.1 cm− 1 almost appears and relates to the stretching vibration mode of a symmetric alkyl C–H C-H. Zein, as previously reported, shows two prominent absorption peaks at ν 1690 and 1550 cm− 1 typical amide I and II bands. The amide I band gradually shifts from ν 1690 to 1618.5 cm− 1 with decreased intensity. While ν 1550 cm− 1 appears at the same wavenumber [37]. Moreover, a long broad peak band at ν 3334 cm− 1 is attributed to O-H stretching vibration and was shifted to ν 3275 cm− 1. The result may indicate that hydrogen bonds between amide groups of glutamines in zein and hydroxyl groups in alginates are formed. The same results were previously obtained with a zein/propylene glycol alginate binary mixture (Fig. 1B) [38].

CuO NPs loaded films (F7-F10) are characterized by increased intensity of absorption peaks in the range of ν 500–700 cm− 1, which reveal the vibrational modes of Cu-O and confirm CuO NPs incorporation. In addition to, its characteristic peaks at ν 1335.06 cm− 1, 1260.94 cm− 1, 3389.82 and 2926 cm− 1, which relate to the stretching of COOH and amino groups absorption peaks, stretching of O-H groups of alcohols and phenols, N-H amines of the amides, in addition to C-H stretching of alkanes, that may be attached to the surface of CuO NPs; respectively, Fig. 1B [39].

SEM investigation

SEM images of crosslinked film formulations F5- F10 at different magnification powers are examined (Figs. 1C and 2). At high magnification, AFs (F5) crosslinked with Ca2+ are observed to be heterogeneous, and dense surface structural with large dents. Upon soaking the film in CaCl2, swelling and polymer folding occur resulting in a lack of surface homogeneity upon further drying (Fig. 2A) [34]. In accordance to Norajit et al. [40], a fibrous-like structure was observed when alginate was crosslinked with Ca2+ contrary to a homogenous plain uncross-linked alginate film. Interestingly, the addition of zein to alginate film, (F6), results in the formation of a particulate system, with a roughly spherical shape and a smaller diameter, adsorbed on the alginate matrix. This can be attributed to the antisolvent precipitation of zein; in addition, the presence of Ca2+ may take a part in crosslinking zein particles. The same finding is observed previously when zein is imaged in the presence and absence of Ca2+ and reported the formation of protein aggregates [38]. On the other hand, the incorporation of CuO in AZFs (F10) is evidenced by the appearance of small discrete crystals of various sizes dispersed throughout the mass of the films and appertaining to the fact that CuO is not water-soluble (Fig. 2A) [6].

Fig. 2

(A) SEM images of samples coded F5, F6, and F10 at high original magnification (10,000x), (B) XRD spectra of F5, F6, and F10; (C) swelling pattern of F5, F6, F7 and F10 in distilled water over 24 h at RT; and (D) % cell viability of plain CuO NPs powder, F7, and F10 at different concentrations (0-0.8 mg/mL) on HBF4 cell line

X-ray diffraction (XRD)

Crosslinked alginate film (F5) exhibits two characteristic peaks at 2θ diffraction angles of 13° and 22°. Additional characteristic peaks for calcium alginate are observed at angles of 13°, 56°, 20.64°, 20.06°, 28.96° as well as 36.40°, revealing its semi-crystalline nature (Fig. 2B) [36]. Incorporation of zein onto alginate film (F6) reveals two broad peaks having maxima at 8.99◦ and 19.38◦ which are in an agreement with Kayaci and Uyar [41] and Sun et al. [38]. It is worth to mention that, compared to the separate patterns of zein and alginate, there is a distinctive new broad peak at the diffraction angles of 30° for zein-alginate bicomplex, which designating the formation of an amorphous complex with the intermolecular interaction occurred between both polymers. The same is observed by Sun et al. [38], when zein is combined with propylene glycol alginate. Meanwhile, the diffraction pattern of CuO NPs-loaded AZFs, F10, makes an evident that CuO maintains its crystalline nature. The diffraction angles are observed to be at 2θ ~ 32.47°, 35.49°, 38.68°, 48.65°, 53.36°, and 58.25° that are assigned to the reflection lines of monoclinic CuO NPs [42].

Moisture uptake (MU%)

Basically, both natural polymers can highly permeate and absorb water. AFs and AZFs are prepared by no or low concentration of CaCl2 as (blending films) show high MU% (Table 2). Crosslinked AFs (F3) shows a 34.62% reduction in MU%, compared to un-crosslinked AFs (F1). The same is observed in crosslinked AZFs (F4), where a 9.92% reduction is obtained, compared to un-crosslinked AZFs (F2).

Furthermore, it is observed that MU% of both Afs and AZFs films is affected greatly by the immersion in 5% CaCl2, dipping films (F5, F6). They show a significant reduction (P > 0.05) by 56.99 and 11.56%; respectively, compared to the corresponding blending films. It is assumed that ionic crosslinking imparted a reduced polymer segmental mobility especially at the surface; thus reducing MU% through the film matrix [6]. The addition of zein to all AFs shows a significant reduction (P < 0.05) in their MU%. This may be attributed to the hydrophobicity of zein creating hydrophobic spots within the hydrophilic matrix of alginate. In addition, it is suggested that water vapor transfer depends mainly on the ratio between the hydrophilic and hydrophobic portions as water vapor permeation occurs only through the hydrophilic portion [40].

Loading films with CuO NPs flaunts a reduction in MU% in all films and reduction increases as concentration of CuO NPs increases. AFs loaded with 0.4% w/v CuO NPs (F7) reveals a 16.7% reduction, compared to unloaded film (F5). While for loaded AZFs (F8-10) reduction of 10.9, 21.05, and 58.57% is obtained upon increasing CuO NPs from 0.1%, w/v to 0.2 and then to 0.4%, w/v. This may be attributed to the tortuous pathway created by impermeable crystalline CuO NPs distributed in the matrix. Thereby they increase the effective diffusion pass length travelled by the water molecules. The same has previously reported when AFs were loaded with cellulose NPs [4, 22]. Since it is recommended for the film to endure an excellent barrier property, and the low permeability of CuO NPs loaded films prove its suitability with enhanced barrier performance.

Swelling and degradation percentage (SW% and D%)

An effectual wound dressing must have the competence of absorbing wound exudates and gradually degrade at a suitable performance to allow a controllable drug release. Both control and blending films AFs and AZFs without CaCl2 treatment are almost utterly dissolved within 5 min after immersion in water, which makes it impossible to measure the SW% (Table 2). It may be attributed to the hydrophilic nature of alginate and no or inadequate crosslinking in the case of blending films. On the other hand, the dipping films retained their integrity for up to 24 h with increases SW% as the immersion time increases (Fig. 2C). The above phenomena is due to the crosslinking activity of CaCl2, renders combined-crosslinked AFs and AZFs insoluble and resist the degradation in water [43].

It is observed that, encompassing zein in F6 film is accompanied by a significant decrease (P < 0.05) in SW and D% of films by 27.4 and 22.04%; respectively, compared to plain alginate film (F5). By taking into account the hydrophobic nature of zein incorporated in preparation of the films, the water degradation resistance increases [10]. It is also worth mentioning that, hydrogen bond formation between the two polymers improves the cohesiveness property of the biopolymer matrix and decreases water aptitude to break this bond [4].

To boot, loading of CuO NPs to films imparted an increase film durability with a significant reduction effect (P < 0.05) on both SW and D% [40]. AFs loaded with CuO NPs (F7) show a decrease in both 56.5% of SW% and 15.7% of D%, compared to unloaded film (F5). AZFs loaded with CuO NPs (F8-10) revealed CuO NPs concentration-dependent reduction in SW%. When CuO NPs increases from 0.1 (F8) to 0.4% (F10), the reduction in SW% increases from 1.36 to 52.26% with an insignificant influence on degradation (Table 2). This reduction in SW% of films may be related to a strong hydrogen bond formation between CuO NPs and the film matrix, attenuating water’s ability to break the bond; thus, SW% decreases [4]. Also, increasing CuO NPs concentration has insignificant (P > 0.05) influence on D%.

From the above results, it can be concluded that as the concentration of CuO NPs increases, the swelling percentages significantly decreases, which is an imperative characteristic that enables both drug release and absorption of excess exudate from the wound. The water absorption causes gradual degradation of the film membrane leading to the sustained release of active pharmaceutical ingredients.

Mechanical properties

Tensile strength (N/mm2), elongation to break (%E) and the elastic modulus (N/mm2) of crosslinked films are depicted in Table 3. The unloaded CaCl2-crosslinked film AF (F5), exhibits a high TS value of 35 ± 3.04 N/mm2, elastic modulus of 2.25 ± 0.19 N/mm2, and elongation percentage of 210 ± 4.73%; which can be correlated to the hydrocolloidal nature of alginate polymer that imparts flexibility and stoutness of alginate films [35]. The inclusion of zein in F6 decreases TS, elastic modulus, and elongation percentage values to 28 ± 1.71 N/mm2, 1.87 ± 0.32 N/mm2, and 171 ± 3.89%; respectively. An explanation for such a phenomenon is the fragility of zein that was previously reported [35]. In fact, zein films are biopolymer of protein origin with a brittle structure while alginate films have more elastic, well-organized, and strong film structures. The brittleness and elasticity disputes in zein films are sequels of strong hydrophobic reactions that stock zein molecules together; however, alginate could be used to reimburse these defects and recuperate its mechanical attributes [35].

Table 3 Mechanical properties of distinct films coded (F5-F10) (mean ± SD, n = 3)

It is observed that, the incorporation of CuO NPs (0.4%, w/v), F7, enhances the mechanical strength of AF. Moreover, increasing the concentrations of CuO NPs incorporated in AZFs results in a significant increase in the ultimate TS values. The TS values of F8, F9 and F10 are 44 ± 2.98, 49 ± 2.54 and 62 ± 3.43 N/mm2; respectively (Table 3). While the elastic modulus of films followed a similar trend to the TS. As the concentration of CuO NPs increases from 0.1 to 0.4%, w/v (F8-F10), the elastic modulus values increase from 3.25 ± 0.35 to 4.15 ± 0.21 N/mm2. This can be accredited to the formation of bonds between hydroxyl groups of the used polymers and CuO NPs. In accordance with previous research work, CuO NPs can play a role in enhancing the stiffness of AZFs by strong interfacial interaction across NPs-polymer matrix [44]. In general, the ultimate TS and elongation at break of biofilms are influenced by NPs distribution throughout the polymer matrix. It seems that the interaction between CuO NPs and the polymer matrix resulted in an increase in TS with reduced films ‘elongation at break, as a result of a decline in the polymer chains mobility. Similarly, in this work, a same decrease in the films’ elongation is observed as the concentration of CuO NPs increases, as shown in Table 3. The same findings was previously obtained where CuO NPs served as anti-plasticizing agents, that can diminish the flexibility of the films by enhancing the interactions and reducing the free spaces between the chains of the biopolymer [45, 46].

Determination of antimicrobial activity of film formulations by disc and well-diffusion method

One of the most serious threats we face as a global community is antimicrobial resistance to common antibiotics [27, 28, 47]. As a result, discovering new antibacterial agents of alternative products with proficient properties in the treatment of these multi-drug resistant human pathogens is a top priority [30]. There have been several antibiotic resistance profiles discovered for human pathogens. As a result, terms including multidrug-resistant, extensively drug-resistant, and pan drug-resistant microbial cells were employed to characterize them [48]. Extensively multi-drug-resistant strains, according to the European Centre for Disease Prevention and Control, that are resistant to at least one antibiotic from three or more antimicrobial classes. On the other hand, multi-drug-resistant bacteria are only sensitive to one or two antibiotic classes, then pan drug-resistant microbial cells refer to microbes that are resistant to all commercially available or routinely tested antibiotics [25, 49]. So, in this work, the selected human pathogens were tested against several antibiotic classes, as indicated in Table 4. In susceptibility testing results, inhibitory zones were classified as having no activity (0 mm, ‘-‘), less activity (1–10 mm, ‘+’), moderately active (11–15 mm, ‘++’), and highly active (16–25 mm, ‘+++’). All examined human pathogens were classified as extensively multi-drug-resistance microbial cells because they were resistant to at least one of the antibiotics tested, as reported in Table 4.

Table 4 Antimicrobial susceptibility test results using different antibiotic categories (which films have been here tested)

To improve metal oxide NPs activity, a variety of protein bases can be employed in the formulation of nano systems. Zein is one of the most frequently utilized protein tools for formulating nano-carriers in the food and pharmaceutical industries, due to its biodegradability, biocompatibility and low cost. Since zein can be used as an eco-friendly carrier system to provide acceptable release qualities and decrease toxicity in the environment [50,51,52]. Herein, zein with alginate are utilized for formulating different concentrations of CuO NPs such as 0.1%, F8 (IV), 0.2%, F9 (V), and 0.4%, F10 (VI). Then, the antagonistic effects of alginate/zein/CuO NPs films are assessed and compared to films that containing alginate, F5 (I), alginate/0.4% w/v CuO NPs, F7 (II), and alginate/zein, F6 (III) against extensively drug-resistant human pathogens using both disc and well diffusion methods (Table 5).

Table 5 Antagonistic efficacy of different films containing different concentrations of CuO nps; 0.1%w/v CuO NPs-loaded AZF, F8 (Treatment IV), 0.2%w/v CuO NPs-loaded AZF, F9 (Treatment V), and 0.4%w/v CuO NPs-loaded AZF, F10 (Treatment VI) were assessed and compared to films that contained only alginate, blank AF F5 (Treatment I), 0.4%w/v CuO NPs-loaded AF, F7 (Treatment II), and blank AZF, F6 (Treatment III) against multidrug-resistant human pathogens using both (D) disc and (W) well diffusion methods

By the disc diffusion method, tested synthetic films (containing CuO NPs) have suppressed the growth of some human pathogens (Fig. 3A). In case of F10 (VI) discs, the largest inhibition zones are obtained against Saccharomyces cerevisiae (21.89 ± 3.59), followed by Escherichia coli (20.12 ± 4.56), and Pseudomonas aeruginosa (18.36 ± 6.23) as in Table 5. However, no inhibitory zones are detected against any of tested Gram + ve bacteria (Fig. 3B). Secondly, via the well diffusion method, we notice that all alginate/zein/CuO NPs samples coded IV, V, and VI, as well as alginate/0.4% CuO NPs (II), suppressed the growth of all examined human pathogens (Fig. 3C). The highest hole zones are determined statistically in case of F10 (VI) against Escherichia coli (25.78 ± 5.96) from Gram-ve bacteria followed by Saccharomyces cerevisiae, (23.79 ± 5.69) from fungal cells and Streptococcus spp. (20.18 ± 3.69) from Gram + ve bacteria (Table 5). Additionally, the samples that did have not CuO NPs (F5, F6) show no inhibition zones by using both methods.

Fig. 3

(A) Antimicrobial activity photographs showing different formed inhibition zone and (B) histogram showing the developed inhibition zone for blank AF (F5), blank AZF (F6), 0.4%w/v CuO NPs-loaded AF (F7), 0.1%w/v CuO NPs-loaded AZF (F8), 0.2%w/v CuO NPs-loaded AZF (F9), and 0.4%w/v CuO NPs-loaded AZF (F10); using disc-diffusion method against multidrug-resistant human pathogens (a) Salmonella paratyphi, (b) Shigella spp., (c) Pseudomonas aeruginosa, (d) Escherichia coli, (e) Streptococcus spp., (f) Staphylococcus epidermidis, (g) Staphylococcus aureus, (h) Candida kruisei, (i) Saccharomyces cerevisiae and (j) Candida albicans. (C) The antagonistic efficacy photographs that indicate different inhibition zones for liquid phase of F5-F10 formulations. (D) Chart showing the developed inhibition zones using well diffusion method of F5-F10 formulations against (a) Salmonella paratyphi, (b) Shigella spp, (c) Pseudomonas aeruginosa, (d) Escherichia coli, (e) Streptococcus spp, (f) Staphylococcus epidermidis, (g) Staphylococcus aureus, (h) Candida kruisei, (i) Saccharomyces cerevisiae and (j) Candida albicans

It can be concluded that, the antimicrobial effect is perceived due to CuO NPs, which can emit reactive oxygen species (ROS); that are responsible for damaging the bacteria’s cells [53]. Moreover, upon increasing CuO NPs concentration from 0.1 to 0.4%, w/v; the antimicrobial potential is significantly enhanced. Meanwhile, antibacterial activity of CuO towards Gram-negative bacteria was superior with regard to Gram-positive ones. The disparity in activity could be appertain to the cell membrane structure and composition. Gram-positive bacteria with a thicker peptidoglycan cell membranes than Gram-negative, make it firmer for CuO to invade it, instigating a lower antibacterial response [42].

Based on these results, we conclude that F10 (VI) formula has an effective antimicrobial activity against extremely drug-resistant human pathogens of both Gram -ve and Gram + ve strains, as well as fungal cells. To detect the effective doses, different dilutions of alginate/zein/ 0.4%w/v CuO NPs (VI) formula are prepared using Milli-Q H2O; such as 10, 30, 50, and 70% (v/v) that are coded as T1, T2, T3, and T4 respectively (Table 5). As can be seen in Figs. 4A-D and 30% (T2), 50% (T3), and 70% (T4) ratios respectively; are significantly increased antagonistic efficacy against all tested human pathogens. The most efficient dilution that form the largest inhibitory zone against all tested bacterial human pathogens is 50% (T3). While the most effective one in the case of fungal cells is 70% (T4). Furthermore, 10% (T1) dilution seems to have the lowest inhibitory zones against all pathogens tested. The highest hole zones are determined statistically bacterial cells; are recorded by using T3 (50%) against Escherichia coli (39.26 ± 3.78) followed by Streptococcus spp. (35.6 ± 5.12). Furthermore, T4 (70%) increased the inhibition zone against fungal cells to 37.94 ± 8.12 in the case of Saccharomyces cerevisiae, followed by Candida albicans (35.19 ± 6.09), and Candida krusei (33.1 ± 8.36) that can be seen in the Table 6.

Table 6 Anti-biofilm efficacy of different dosages of formulations against different multidrug-resistant human pathogens in-vitroFig. 4

(A) Antagonist efficacy of dilute 0.4%w/v CuO NPs-loaded AZF (F10) formulation, (T1; 10% V/V), (T2; 30%V/V), (T3; 50% V/V) and (T4; 70% V/V) against (a) Salmonella paratyphi, (b) Shigella spp., (c) Pseudomonas aeruginosa, (d) Escherichia coli, (e) Streptococcus spp., (f) Staphylococcus epidermidis, (g) Staphylococcus aureus, (h) Candida kruisei, (i) Saccharomyces cerevisiae and (j) Candida albicans. These photo point to inhibition zones of dilute F10 formulation against (B) Escherichia coli, (C) Streptococcus spp., and (D) Saccharomyces cerevisiae. The effectiveness and durability of antimicrobial activities of dilute F10 formulation (50%) stored at (E) 4°C and (F) 30°C against Escherichia coli, Streptococcus spp., and Saccharomyces cerevisiae

The antibacterial efficiency and durability of diluted F10 formula (50%) are tested against Escherichia coli, Streptococcus spp., and Saccharomyces cerevisiae by packing this formula at 4 °C and 30 °C, and inhibited zones are recorded at intervals. The baseline zones of inhibition diameters (0 day) are estimated after tested composition; is prepared. According to displayed data, antimicrobial activities of this formula continued for 30 days in both cases (Fig. 4E and F). Furthermore, within 15 days, antimicrobial activities of tested formula at 4 °C and 30 °C are nearly the same against all tested strains (p > 0.05), suggesting that CuO NPs are released continuously from AZFs. Interestingly, the examined formula’s antimicrobial activity held steady for 30 days after being stored at 4 °C. The antimicrobial activity of the formula stored at 30 °C appear the lowest over 15-to-30-day period. During this period (15 days), the release of CuO NPs from F10 formulation (50%) exhibits a sustained antimicrobial activity, which may just protect cutaneous wounds from infection. According to this data, once this period has passed, there is no longer a risk to human health. As an outcome of this study, a sustained-release CuO NPs from AZFs (F10) at 50% concentration, results in faster wound healing which will aid to reduce the price of burns treatment by allowing for an earlier hospital discharge. As a result, this formula is expected to become a valuable wound and burns therapy prefect tool in the future.

In vitro cell viability

The cytotoxicity of CuO NPs loaded AFs (F7), AZFs (F10), as well as CuO NPs on HBF4 cells over the range (0-0.8%) are tested (Fig. 2D). Both films exhibited higher safety on the viability of HBF4 cells than free CuO NPs. The estimated IC50 values are 1.002 ± 0.05%, 1.487 ± 0.06%, and 1.933 ± 0.05% for free CuO NPs, CuO NPs AFs (F7), and CuO NPs-loaded AZFs (F10); respectively. Additionally, the safe doses (EC100) are 0.071 ± 0.001%, 0.097 ± 0.007%, and 0.121 ± 0.001%; respectively. This indicates the highest IC50 and EC100 values of CuO NPs-loaded AZFs (F10), compared to AFs and free-CuO NPs.

Zein proposes extra potential benefits as a cyto-compatible protein for proliferation of NIH3T3 cells as well as HL-7702 cells [9]. It is noticed that cell viability is dependent on the concentration of loaded-CuO NPs, where cell viability decreases with increasing concentration. Lipid peroxidation and oxidative stress have been previously reported to be one of the toxicity mechanisms related to CuO NPs exposure [54]. It is also noticed that cell viability of 0.4% w/v loaded AZFs (F10) ranged from 60 to 89%. This may be attributed to its green nature synthesis, where CuO NPs are coated with capping and stabilizing agents from the plant extract which enhancing its biocompatibility. The same is observed when CuO NPs are previously coated with biopolymers, such as hyaluronic acid. The obtained results for cell viability show that ions like Cu2+ can be deliberated as safe ions for biomaterial application specially at low doses [55].

In vivo wound healing potencyWound healing appraisal

Alginate and zein could have the capability to synergistically accelerate the diabetic wound healing process in the rat model. Both polymers can act as reservoirs for active agents, biocompatible, and biodegradable. Alginate has a hemostatic property, while zein has antimicrobial and antioxidant properties. The diabetic wound healing potential of the biopolymers was tested on circular wounds on the dorsal area of diabetic rats [6, 14, 56].

Principally, copper, has a crucial starring role in wound restoration by stimulating angiogenesis, and enhancing cell migration with proven wide spectrum bactericidal effect claiming for copper oxides application to be convenient, but at the same time risky [21, 57, 58]. In this regard, the incorporation of CuO NPs into AZF is intended to control the release of CuO NPs, due to the presence of hydrophobic polymer like zein and CaCl2 as a crosslinker. Such a retardation in the released amount would reduces the cytotoxicity and enhances th