STRUCTURAL AND ANTIMICROBIAL PERFORMANCE OF POLYVINYL ALCOHOL (PVA) BIOFILMS REINFORCED WITH CUO MICROPARTICLES SYNTHESIZED VIA A SIMPLE CO-PRECIPITATION ROUTE

Abstract

This study investigates the influence of copper oxide (CuO) microparticles with different morphologies on the structural, mechanical, and antimicrobial properties of polyvinyl alcohol (PVA) biofilms. CuO microparticles were synthesized via a simple co-precipitation method under controlled pH and thermal conditions, yielding fibrous, granular, and spherical morphologies. The particles were physically dispersed into the PVA matrix at different weight ratios using the casting method. The resulting composites were characterized by X-ray diffraction (XRD) with Rietveld refinement, dynamic light scattering (DLS), Fourier-transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), mechanical testing, and microbiological assays. The results demonstrate that both particle morphology and concentration significantly affect film crystallinity, Young’s modulus, and tensile strength. Fibrous CuO microparticles enhanced surface antimicrobial activity, producing larger inhibition zones against Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923, whereas spherical particles exhibited superior bactericidal performance in liquid culture due to improved dispersion and sustained Cu²⁺ ion release. Despite the absence of particle functionalization, which may have limited interfacial interactions, the composites exhibited promising multifunctional performance. These findings highlight the critical role of particle shape, distribution, and interfacial quality in tailoring the properties of PVA/CuO biofilms.

Keywords:
antimicrobial activity; CuO microparticles; polymeric films; PVA films.

INTRODUCTION

In recent years, polymeric films with antimicrobial properties have emerged as promising alternatives for biomedical applications, active packaging, and protective barriers in contaminated environments. Among the most widely used polymers, polyvinyl alcohol (PVA) stands out due to its biocompatibility, water solubility, low cost, and ease of structural modification.

The incorporation of metallic particles into PVA has been extensively associated with enhanced mechanical properties, such as increased tensile strength and elastic modulus.¹ These improvements are attributed to the formation of effective interfaces that facilitate load transfer between the dispersed phase and the polymer matrix. In addition to structural reinforcement, these particles confer multifunctional properties to the composites, including improved thermal stability, reduced gas permeability, electrical conductivity, and antimicrobial activity.²-⁴

Among the inorganic additives investigated, copper oxide (CuO) is particularly notable for its well-documented antimicrobial activity, primarily attributed to the release of Cu²⁺ ions and the generation of reactive oxygen species.⁵-⁸ While CuO nanoparticles are widely studied for their high efficacy, the use of microparticles presents a promising alternative. Microparticles offer advantages such as lower toxicity, improved biocompatibility, and ease of synthesis through simple and cost-effective methods like co-precipitation followed by thermal treatment,⁹-¹² eliminating the need for complex size control steps and contributing to a more sustainable and reproducible process at the laboratory scale.¹³

Despite the growing body of literature on antimicrobial films, systematic investigations into the influence of CuO particle morphology on the antimicrobial mechanism under different exposure conditions remain limited. In this context, the present study aims to address this gap by evaluating the performance of PVA films reinforced with CuO microparticles synthesized via a simple co-precipitation method,¹⁴,¹⁵ resulting in distinct particle morphologies.

The antibacterial activity of the developed materials was evaluated using Escherichia coli and Staphylococcus aureus as representative microorganisms, selected due to their contrasting cell wall architectures and widespread relevance in antimicrobial assessments. E. coli, a Gram-negative bacterium, possesses an outer membrane that acts as an additional barrier to antimicrobial agents, while S. aureus, a Gram-positive bacterium, is characterized by a thick peptidoglycan layer and is a common pathogen in both clinical and environmental settings. The use of these two strains enables a comprehensive evaluation of antibacterial efficacy against microorganisms with distinct structural and physiological features.¹⁶,¹⁷

This investigation adopts a multidisciplinary approach, encompassing structural, morphological, mechanical, and antimicrobial analyses. Characterization techniques employed include X-ray diffraction (XRD) with Rietveld refinement, dynamic light scattering (DLS), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and mechanical testing. These analyses enabled the assessment of particle dispersion within the matrix, interfacial interactions, and their effects on film crystallinity and mechanical performance.¹⁵-¹⁸ The antimicrobial activity of the composites was also evaluated, with a focus on the potential of CuO microparticles to inhibit microbial growth through the controlled release of metal ions, using Escherichia coli and Staphylococcus aureus as model organisms.

EXPERIMENTAL

Synthesis of CuO microparticles

CuO particles were synthesized via the co-precipitation method, following specific conditions for each type:

Type D particles were prepared using copper(II) sulfate pentahydrate (CuSO₄·5H₂O, 98%, Dinâmica) at a concentration of 1 mol L^-1 as the copper source. A 2 mol L^-1 sodium hydroxide (NaOH, Dinâmica) solution was added dropwise under continuous stirring. The resulting suspension was left to rest at room temperature for 12 h to allow precipitate formation. The solid was then washed with distilled water, dried in an oven at 80 °C for 6 h, ground, and calcined in a muffle furnace at 350 °C for 2 h under an oxidative atmosphere.¹⁹

Type E particles were also synthesized by co-precipitation, using copper(II) nitrate trihydrate (Cu(NO₃)₂·3H₂O, 1% w/w, Dinâmica) as the metal precursor and 1 mol L^-1 NaOH as the precipitating agent. The base was added gradually until the pH reached 10, maintaining continuous magnetic stirring for 3 h at room temperature. The resulting solid was washed with distilled water, dried at 130 °C for 10 h, ground, and calcined at 350 °C for 2 h in an oxidative atmosphere.¹⁸

Type G particles were synthesized using the same method, but under neutral pH conditions. Initially, 5 mmol of Cu(NO₃)₂·3H₂O were dissolved in distilled water and reacted with 10 mmol of NaOH, adjusting the pH to 7. The mixture was stirred for 1 h and then left undisturbed at room temperature. The precipitate was separated by centrifugation, washed with ethanol, air-dried, ground, and finally calcined at 350 °C for 2 h in an oxidative atmosphere.¹⁸

Table 1 presents a comparative summary of the main synthesis parameters used for the preparation of D, E, and G type particles, highlighting the specific characteristics of each protocol, including the precipitation, drying, and calcination conditions employed.

Table 1
Synthesis parameters used for the preparation of CuO microparticles with different morphologies (D, E, and G types), including variations in precursor salts, precipitating agents, pH conditions, reaction times, washing procedures, drying methods, and thermal treatments

Parameter	D type	E type	G type
Precursor salt	CuSO4·5H2O (1 mol L-1)	Cu(NO3)2·3H2O (1% p/p)	Cu(NO3)2·3H2O (5 mmol)
Precipitating agent	NaOH (2 mol L-1)	NaOH (1 mol L-1)	NaOH (10 mmol)
Final pH	not adjusted	pH 10	pH 7
Stirring time	during addition	3 h	1 h
Resting time	12 h	-	after stirring
Washing	distilled water	distilled water	ethanol
Drying	80 °C, 6 h	130 °C, 10 h	air drying
Thermal treatment	350 °C for 2 h	350 °C for 2 h	350 °C for 2 h

Preparation of PVA films reinforced with CuO particles

PVA films reinforced with CuO particles of different morphologies were prepared using the casting technique, employing three distinct weight ratios of filler, classified as series D, E, and G. To prepare the polymer solution, 5 g of PVA were dissolved in 100 mL of distilled water at 60 °C under constant stirring until complete homogenization was achieved. CuO particles were then incorporated into the solution at concentrations of 1, 5, and 10% by weight relative to the polymer. During the dispersion process, the temperature of the solution was gradually increased to 75 °C and maintained for 30 min. Subsequently, 5 g of glycerol were added as a plasticizer, and the mixture was kept under the same temperature and stirring conditions for an additional 30 min. After this period, the solution was gradually cooled under continuous stirring and poured into Petri dishes. The films were dried in an oven at 60 °C for 24 h.

We emphasize that the CuO particles were used without any type of surface functionalization. Although this simplified the fabrication route and allowed us to observe the morphology effects in person, we acknowledge that the absence of chemical modification may have limited interfacial adhesion and homogeneous dispersion within the polymer matrix, especially at higher concentrations. The concentrations of microparticulate CuO (1, 5, and 10% by weight) used in the preparation of the films were chosen based on preliminary studies not included in this manuscript, in which good processability and formation of homogeneous films were observed within this range.

Characterization of CuO and PVA/CuO films

X-ray diffraction

XRD measurements were performed using a Bruker D2 Phaser diffractometer (Bruker AXS, Karlsruhe, Germany) equipped with a Cu Kα radiation source, with a wavelength (λ) of 0.15406 nm. The diffraction patterns were refined using the Rietveld method with GSAS-EXPGUI software,²⁰ allowing us to obtain crystallographic information such as lattice parameters, unit cell volume, phase percentages, and average crystallite size. Peak profile fitting was accomplished using the modified Thompson-Cox-Hastings pseudo-Voigt function, which is included in the GSAS package.²¹ The average crystallite size (t) was calculated using Equation 1:

(1) $$\mathrm{t}=\frac{18000\mathrm{K}\lambda }{\pi \mathrm{X}}$$

where X represents the isotropic broadening due to crystallite size and K is the Scherrer constant (0.91). The degree of crystallinity (χ_C) was calculated using Equation 2:

(2) $${\chi }_{\mathrm{C}}(\mathrm{\%})=\frac{{\mathrm{I}}_{\text{total}}-{\mathrm{I}}_{\mathrm{BG}}}{{\mathrm{I}}_{\text{total}}}\times 100$$

where I_total is the total intensity of the crystalline peaks and I_BG is the background scattering simulated using a 12-term Chebyshev polynomial to represent the amorphous phase, following the methodology previously described.²²

Fourier transform infrared spectroscopy

Infrared spectroscopy analyses were performed using a Bruker FTIR spectrometer, model Vertex 70V, operating in attenuated total reflectance (ATR) mode. The CuO samples were analyzed directly on the ATR crystal without the need for additional preparation. Spectra were collected over the wavenumber range of 4000 to 40 cm^-1, with a resolution of 4 cm^-1 and an average of 32 scans per measurement.

Dynamic light scattering (DLS)

The particle size distribution of CuO particles (types D, E, and G) dispersed in an aqueous medium was analyzed using a HORIBA SZ-100 nanoparticle analyzer. This technique provided information on the hydrodynamic diameter and average size of the particles in suspension. Additionally, the same instrument was used to measure the zeta potential of the particles, giving insights into their surface charge and colloidal stability in aqueous solutions.

Scanning electron microscopy (SEM)

SEM images were obtained using a high-resolution scanning electron microscope (JEOL JSM IT500-HR). The samples were fixed onto stubs with carbon tape and analyzed without any metallic coating.

Mechanical tests of PVA/CuO films

Tensile tests were conducted using a universal testing machine (Técnico Industrial Oswaldo Filizola Ltda, model AME-5kN). The films were cut according to ASTM D882-02²³ standards for thin plastic films, in order to determine tensile strength and Young’s modulus. The tests were performed at room temperature at a speed of 5 mm min^-1, and each condition was tested in triplicate. The specimens had dimensions of 10 mm × ε mm × 30 mm (width × thickness × length), with the thickness measured using a digital micrometer.

Microbiological assays

Antimicrobial activity of PVA/CuO films

The antimicrobial activity of polymeric films was evaluated using the agar disk diffusion method, adapted from Bauer et al.²⁴ and in accordance with the NCCLS guidelines,²⁵ 1 mL bacterial inocula (3 × 10⁸ UFC mL^-1) strains Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923, were plated onto Mueller-Hinton agar plates (HIMEDIA®), and film discs (4 mm in diameter) were carefully placed on the surface. After standing at room temperature for 30 min, the plates were incubated at 36 ± 2 °C for 24 h. At the end of the incubation period, the diameter of the inhibition zone was measured in millimeters. All tests were performed in triplicate.

Minimum inhibitory concentration (MIC)

The MIC assay for CuO particles (types D, E, and G) was conducted using stock solutions prepared at a concentration of 5 mg mL^-1 in autoclaved distilled water. The negative control consisted solely of the vehicle solution, while the positive control used ampicillin at the same concentration, appropriate for the bacterial strains E. coli and S. aureus. In the initial screening, 96 well microplates were used, where 100 µL of double-strength culture medium, 100 µL of the test sample (2.5 mg mL^-1), and 10 µL of bacterial suspension, adjusted to 0.5 on the McFarland scale (1.5 × 10⁸ CFU mL^-1), was added to each well in triplicate. After incubation at 36 ± 1 °C for 24 h, 10 µL of 1% TTC (2,3,5-triphenyltetrazolium chloride) solution was added to detect microbial growth, indicated by the development of a pink color. As a counter-check, 10 µL from each well were plated on solid medium. Samples that demonstrated antimicrobial activity underwent serial dilutions of 1:2, transferring 100 µL of culture medium and 100 µL of the sample sequentially from wells in row A to row G of the plate. The wells in row H were left as growth control (Co). Again, 10 µL of bacterial suspension was added to all wells, with the controls following the same protocol. The MIC was defined as the lowest concentration that did not result in a color change, indicating the absence of bacterial growth.²⁶

Colony forming unit (CFU) count

The antibacterial activity of CuO microparticles was evaluated in isolation, in the absence of any polymeric matrix (PVA), with the aim of exclusively investigating the intrinsic antimicrobial effect of copper oxide. For this purpose, a CFU counting assay was performed to quantify bacterial viability after treatment with CuO suspensions.

Initially, CuO microparticles of types D, E, and G were individually dispersed in sterile aqueous medium at a concentration of 2.5 mg mL^-1, previously determined as the MIC. The suspensions were subjected to vigorous stirring and ultrasonication to ensure proper particle dispersion and to minimize agglomeration.

Bacterial suspensions were prepared from fresh cultures and diluted in sterile culture medium to an approximate concentration of 10⁷ CFU mL^-1. These suspensions were then incubated with solutions containing exclusively the CuO microparticles for 24 h at 37 °C under controlled conditions. Control assays were conducted simultaneously using only the bacterial suspension in sterile medium, without the addition of CuO, allowing direct comparison of microbial viability.

After the incubation period, serial decimal dilutions of the treated suspensions were prepared. Aliquots of 100 µL from each dilution were spread onto Petri dishes containing Mueller-Hinton agar and incubated at 37 °C for an additional 24 h. After incubation, visible colonies were manually counted, with each colony considered a CFU. Bacterial growth inhibition was determined by the reduction in CFU number relative to the negative control, enabling quantification of the viable cells remaining after treatment with the CuO microparticles.

RESULTS AND DISCUSSION

Characterization of CuO particles

The XRD patterns of the synthesized particles display characteristic diffraction peaks of the monoclinic CuO phase, identified according to ICSD card No. 291390, confirming the formation of the desired crystalline structure. This phase corresponds to the thermodynamically stable form of CuO at room temperature, indicating the effectiveness of the synthesis routes employed. The diffraction patterns of the D, E, and G particles were refined using the Rietveld method, and the corresponding figures (Figures 1a, 1c, and 1e) explicitly present the indexed diffraction peaks, with the Miller indices (hkl) associated with the identified crystalline phases clearly indicated.

Figure 1
XRD patterns (black line) and corresponding Rietveld refinements (red line) of the particles: (a) D, (c) E, and (e) G. SEM images obtained for the particles: (b) D, (d) E, and (f) G

The Rietveld refinements showed excellent agreement between the experimental data and the calculated profiles, as evidenced by near-zero residual curves and low values of quality factors, indicating a good fit. Table 2 presents the refined lattice parameters, unit cell volumes, and phase weight percentages for the XRD patterns of particles D, E, and G.

Table 2
Phase percentage, lattice parameters (a, b, c), unit cell volume (V), average crystallite size (t), degree of crystallinity (χ_C), and reliability factors (wRp, χ²) obtained from Rietveld refinement of the XRD patterns

Sample	Phase	Phase / %	a / Å	b / Å	c / Å	V / Å3	t / nm	χC / %	wRp	χ2
Particle D	CuO	100	4.68(4)	3.42(2)	5.12(3)	81.1(8)	282.1	100	0.1553	1.834
Particle E	CuO	93.7	4.68(3)	3.42(3)	5.12(2)	81.1(5)	125.4	100	0.1581	1.906
	NaNO3	6.3	5.06(2)	5.06(1)	16.84(3)	374.6(8)	404.4
Particle G	CuO	100	4.68(2)	3.42(3)	5.12(3)	81.1(4)	168.5	100	0.1556	1.804
Film PVA	PVA	100	7.84(1)	2.52(8)	5.54(6)	109.8(4)	10.1	23.78	0.1062	1.084
Film D1	CuO	62.2	4.78(1)	3.60(4)	4.94(5)	81.1(1)	33.1	7.86	0.0257	1.535
	PVA	37.8	8.21(1)	2.50(7)	5.43(3)	111.7(1)	22.1
Film D5	CuO	5.4	4.67(6)	3.41(3)	5.12(5)	80.7(7)	79.9	11.91	0.0250	1.983
	PVA	94.6	15.07(2)	2.59(4)	4.77(1)	186.5(4)	49.5
Film D10	CuO	55.1	4.67(8)	3.43(3)	5.12(9)	81.0(3)	47.2	12.31	0.0248	1.981
	PVA	44.9	7.87(6)	2.93(2)	5.41(4)	127.0(1)	18.5
Film E1	CuO	15.4	4.71(1)	3.42(1)	5.17(9)	82.7(4)	78.6	6.74	0.0280	1.957
	PVA	84.6	8.96(6)	2.45(2)	5.15(4)	112.8(1)	20.1
Film E5	CuO	48.7	4.69(1)	3.49(4)	5.20(1)	84.1(2)	121.2	6.23	0.0273	1.682
	PVA	51.3	8.74(1)	2.47(2)	5.20(3)	112.1(1)	11.1
Film E10	CuO	11.8	4.70(9)	3.48(4)	5.11(9)	82.7(1)	185.7	20.46	0.0269	1.945
	PVA	88.2	7.98(1)	2.60(5)	6.61(1)	135.0(3)	10.4
Film G1	CuO	5.5	4.71(3)	3.49(1)	5.06(1)	82.23(4)	157.1	8.9	0.0250	1.983
	PVA	94.5	8.24(1)	2.46(3)	5.57(4)	113.3(1)	13.3
Film G5	CuO	1.9	4.79(4)	3.21(1)	5.49(2)	83.3(1)	158.2	10.45	0.0243	1.979
	PVA	98.1	8.21(3)	2.56(2)	5.71(4)	120.1(2)	13.7
Film G10	CuO	23.4	4.69(7)	3.44(6)	5.13(2)	81.6(1)	100.4	9.80	0.0254	1.965
	PVA	76.6	7.83(7)	2.59(2)	5.60(5)	114.0(1)	26.6

It is noteworthy that the diffraction pattern of E-type particles displayed an additional peak at 2θ = 29.32°, marked with an asterisk, corresponding to a secondary phase identified as sodium nitrate (NaNO₃), according to ICSD card No. 64865. This phase, estimated at 6.3 wt.% through Rietveld refinement, is associated with the use of copper nitrate under highly basic conditions, which may have favored the formation and retention of residual salts. In contrast, the diffraction patterns of D and G type particles showed no secondary peaks, indicating high phase purity.

SEM micrographs of the CuO particles, shown in Figures 1b, 1d, and 1f, reveal the direct influence of synthesis conditions on particle morphology. Although all samples were produced using the co-precipitation method, variations in precursor salt, pH, and base concentration led to distinctly different morphologies.

D-type particles, shown in Figure 1b, exhibit a fibrous and branched morphology, attributed to directional growth induced by the highly alkaline synthesis conditions (2 mol L^-1 NaOH). The basic environment favors anisotropic nucleation mechanisms, resulting in the formation of elongated structures, as also reported by Sudha et al.²⁷ The fibers displayed lengths ranging from 0.5 to 2 µm, with a well-defined preferential orientation.

The schematic representation in the inset of Figure 1b supports these observations, highlighting sharp and elongated tips consistent with anisotropic growth. This type of structural organization tends to increase the available surface area, which may enhance interactions with the polymer matrix and promote the release of Cu²⁺ ions - an important feature for antimicrobial applications.²⁸

E-type particles, shown in Figure 1d, display a granular and irregular morphology, with a broad size distribution. This heterogeneity may result from rapid nucleation under pH 10 and a lack of kinetic control during crystal growth, leading to poorly defined edges and low morphological uniformity, as also illustrated in the schematic inset. Such behavior is consistent with the findings of Gholami and Maddahfar,²⁹ who associated uncontrolled growth with irregular morphology and reduced specific surface area, thus impairing particle dispersion within polymeric matrices.

G type particles (Figure 1f) are characterized by a spherical morphology with more homogeneous size distribution and smooth contours, as shown in the corresponding schematic representation. This morphology suggests that the neutral pH and milder precipitation conditions favored isotropic and well-controlled crystal growth. These findings are in agreement with those of Jellicoe et al.,³⁰ who demonstrated that pH control is critical for obtaining spherical particles with high surface area and reduced agglomeration tendency.³¹

Taken together, the results demonstrate that subtle changes in synthesis parameters influence not only the shape and size of the particles but also their surface properties, reactivity, and technological applicability.²⁷-³²

Figure 2 presents the particle size distribution curves obtained by DLS for samples D, E, and G. In Figure 2a, the black curve represents the frequency distribution of D-type particles, revealing a broad size range from 0.1 to 5 µm with a bimodal profile.³³ This indicates the presence of multiple particle populations, likely due to partial agglomeration and the fibrous nature observed in SEM micrographs.³⁴ The corresponding cumulative curve (cyan) shows a gradual slope, further supporting the heterogeneous size distribution.

Figure 2
Particle size distribution curves obtained by DLS for CuO particles of types (a) D, (c) E, and (e) G, accompanied by the corresponding zeta potential distributions shown in figures (b), (d), and (f)

In contrast, Figure 2b shows a unimodal size distribution for E-type particles, with a dominant peak between 0.8 and 1.2 µm. The cumulative curve (yellow-green) rises steeply, reflecting greater uniformity in particle size and reduced agglomeration. Such behavior is commonly associated with improved colloidal stability and more consistent dispersion in suspension.³⁵,³⁶

Figure 2c illustrates the distribution of G-type particles, which exhibit a narrow size range centered around 0.15 µm. The steep and nearly vertical cumulative curve (red) confirms a highly monodisperse system, an essential characteristic for applications that demand precise control over particle dimensions.³⁷ This high level of uniformity contributes to predictable behavior in solution and may enhance the surface reactivity of the particles.³⁸

To complement the particle size analysis and assess suspension stability, zeta potential measurements were performed (Figures 2b, 2d, and 2f). The recorded values were -17.11 mV for D-type particles, -52.78 mV for E-type particles, and -41.67 mV for G-type particles. These values indicate the presence of electrostatic repulsion between particles, a key factor for maintaining colloidal stability in aqueous systems.³⁹

The lower zeta potential of D-type particles aligns with their broader and more heterogeneous size distribution, suggesting a greater tendency toward flocculation over time. In contrast, the more negative zeta potentials of E and G-type particles reflect in stronger interparticle repulsion and, consequently, greater dispersion stability, exhibiting the most favorable characteristics for stable suspension-based applications.⁴⁰

Characterization of PVA/CuO films

XRD and Rietveld refinement analysis

Figure 3a displays the XRD pattern of the pure PVA film (blue line), in which a broad diffraction peak centered around 2θ ≈ 19.6° is observed. This peak is characteristic of its semicrystalline structure and is associated with the partial ordering of the polymer chains.⁴¹ The calculated degree of crystallinity was 23.78%, confirming the semi-ordered nature of the pure matrix.

Figure 3
X-ray diffraction patterns of pure PVA films and composites incorporating CuO particles of types: (a) D, (c) E, and (e) G. Corresponding FTIR spectra of the films containing particles of types: (b) D, (d) E, and (f) G

After the incorporation of CuO particles, all composite films exhibited a progressive reduction in both peak intensity and crystallinity, regardless of particle type. This trend is attributed to the disruption of the PVA chain arrangement caused by the presence of dispersed inorganic fillers. Figure 3a shows this effect for films prepared with type D particles, while similar behavior is observed in Figures 3c and 3e for films containing type E and G particles, respectively.⁴²,⁴³ Notably, an exception was found for the G10 film, which exhibited a slight increase in crystallinity, suggesting that under certain conditions, a higher CuO content may promote polymer chain realignment and structural reorganization.⁴⁴

Rietveld refinements confirmed these structural changes, yielding good fit parameters (wRp (weighted profile R-factor) < 10%, χ² < 2), and further corroborated the variations observed in crystal size and phase distribution. A reduction in the average crystal size of the PVA phase was observed in most composites, indicating stronger interactions between the CuO particles and the polymer matrix. These interactions are likely mediated by hydrogen bonding or coordination between the CuO surface and the hydroxyl groups of PVA.⁴⁵,⁴⁶

For type E films, the refinement results revealed differences in phase composition: E5 exhibited a more balanced distribution of crystalline phases, whereas E10 showed higher overall crystallinity despite a lower amount of detectable crystalline CuO. This suggests that the phase distribution and CuO dispersion directly influence the structural organization of the polymer.⁹,⁴³

In films containing type G particles, a reduction in the crystallite size of the CuO phase was observed with increasing filler content, implying that the polymer matrix affects the crystalline morphology of the inorganic phase.⁴⁷,⁴⁸ Interestingly, the G10 film exhibited an increase in the crystallite size of the PVA phase, which may be attributed to improved packing induced by the higher particle concentration. As reported by Miyazaki et al.,⁴⁷ this phenomenon may reflect enhanced structural compatibility between the polymer and the dispersed phase.

FTIR spectroscopy and interfacial interactions

Figures 3b, 3d, and 3f show the FTIR spectra of films prepared with the dispersion of type D, E, and G particles. All samples exhibit a broad band between 3200-3500 cm^-1, associated with O-H stretching vibrations from hydroxyl groups in the PVA matrix. A gradual decrease in the intensity of this band with increasing CuO content suggests potential interactions between hydroxyl groups and CuO surfaces, possibly through hydrogen bonding. This supports the hypothesis of chemical interaction affecting molecular packing and crystallinity.⁴⁹

Typical vibrational features of PVA are observed in all spectra, such as the symmetric and asymmetric stretching of -CH₂ between 2850-2940 cm^-1, and C-O and C-C stretching modes between 1140 and 1080 cm^-1, confirming the structural integrity of the polymer network.⁵⁰ The peak at 1650 cm^-1, attributed to C=C stretching, may indicate the presence of oxidation products or impurities,⁵¹ while the carbonyl peak at 1730 cm^-1 is assigned to acetate residues - commonly observed in partially hydrolyzed PVA systems.⁵⁰,⁵¹

At lower wavenumbers, in regions corresponding to Cu-O vibrations (below ca. 600 cm^-1), changes in band intensity provide further insights into filler dispersion. In films from the D and E series, a progressive increase in Cu-O band intensity with increasing CuO content suggests better incorporation and matrix-particle interaction.⁵² In contrast, the G1 and G5 films exhibit weak Cu-O signatures, indicating limited dispersion. However, G10 shows a sharp increase in intensity, which may result from particle agglomeration or overload within the matrix - a scenario that, according to Kudo et al.,⁵³ can reduce functional efficiency due to poor interfacial contact.⁵²-⁵⁴

Mechanical properties of PVA/CuO films

The stress-strain curves obtained from tensile tests demonstrate that the incorporation of CuO microparticles significantly influences the mechanical behavior of PVA films. Figure 4 presents the stress-strain curves of the composite films, including neat PVA, which serves as a reference for evaluating the effect of particulate reinforcement on the mechanical performance of the modified systems.

Figure 4
Stress-strain curves of pure PVA and PVA/CuO composite films containing different proportions of D (a), E (b), and G (c)

As shown in Figures 4a-4c, films from the D, E, and G series exhibit concentration-dependent mechanical responses. From these curves, Young’s modulus was determined, providing direct information on the stiffness and elastic behavior of the films.⁵⁵,⁵⁶ These mechanical parameters are closely related to the efficiency of particle-matrix interactions,⁵⁷ which play a crucial role in stress transfer and in the development of well-reinforced polymer composites.

The mechanical properties of the D-series films, presented in Table 3, demonstrate a strong dependence on particle concentration, taking into account the mean values and their respective experimental uncertainties, which reinforces the reliability of the observed trends. The D1 film exhibited the highest stiffness within this series, with a Young’s modulus of 161.9(2) MPa. In contrast, the D5 and D10 films showed lower moduli of 90.6(3) and 105.9(2) MPa, respectively, indicating a reduction in stiffness at higher filler loadings. This behavior suggests increased polymer chain mobility, possibly associated with particle agglomeration at elevated concentrations. The tensile strength of the D-series films varied within a relatively narrow range (18.5(3)-22.0(2) MPa), indicating a balance between stiffness and mechanical resistance across the different compositions.

Table 3
Young’s modulus and tensile strength of pure PVA film and PVA/CuO composite films containing CuO microparticles with different morphologies (D, E, and G) and loadings

Film	Yuong’s modulus / MPa	Tensile strength / MPa
PVA	121.1(4)	30.17(2)
D1	161.9(2)	21.3(8)
D5	90.6(3)	22.0(2)
D10	105.9(2)	18.5(3)
E1	82.2(6)	12.6(5)
E5	96.2(5)	17.9(3)
E10	85.1(3)	16.9(4)
G1	176.9(2)	25.0(2)
G5	168.9(4)	29.8(1)
G10	128.0(2)	22.3(3)

The E-series films exhibited comparatively stable mechanical behavior with increasing particle concentration. Young’s modulus ranged from 82.2(6) MPa for E1 to 96.2(5) MPa for E5, followed by a slight decrease to 85.1(3) MPa for E10. Among these formulations, the E5 film showed the best mechanical performance within this series, combining the highest stiffness with a tensile strength of 17.9(3) MPa. The relatively constant stiffness values suggest that the polymer matrix can accommodate the dispersed E-type particles without significant structural degradation,⁵⁸ although the reinforcement effect is less pronounced than that observed for the other particle morphologies.⁵⁹,⁶⁰

In contrast, the G-series films exhibited a distinct mechanical response. The G1 film presented the highest Young’s modulus among all evaluated formulations, reaching 176.9(2) MPa, together with a tensile strength of 25.0(2) MPa. Notably, the G5 film demonstrated the best overall mechanical performance, combining a high Young’s modulus of 168.9(4) MPa with the highest tensile strength observed (29.8(1) MPa). These results indicate more efficient interfacial interactions between G-type particles and the PVA matrix, promoting improved stress transfer within the composite. For the G10 film, a reduction in stiffness was observed, with a Young’s modulus of 128.0(2) MPa, close to that of neat PVA (121.1(4) MPa), along with a tensile strength of 22.3(3) MPa. This behavior suggests reduced reinforcement efficiency at higher particle concentrations.⁶¹

This behavior is consistent with literature reports indicating that lower concentrations of metallic particles can preserve the toughness of the polymer matrix while still enhancing mechanical strength.⁶² Consequently, formulations with intermediate filler content tend to exhibit a more balanced mechanical performance. Similar trends have been observed in composite systems where a trade-off between stiffness and tensile strength occurs, depending on the nature and concentration of the incorporated fillers.⁶³ For film G10, which exhibited the lowest stiffness in the G-series at 128.0(2) MPa, the data support the hypothesis that both the amount and morphology of metallic particles directly influence the mechanical properties of the PVA-based films.⁶⁴

Antimicrobial activity of PVA/CuO films

The antimicrobial activity of PVA films incorporated with CuO microparticles was evaluated against Escherichia coli (Gram-negative bacterium) and Staphylococcus aureus (Gram-positive bacterium) using agar diffusion, minimum inhibitory concentration (MIC), and colony-forming unit (CFU) assays.

Disk diffusion assay

Figure 5 presents the results of the disk diffusion assay, showing that films from the D and E series exhibited visible inhibition zones against Escherichia coli and Staphylococcus aureus, particularly at higher CuO concentrations (10%). The D-series films, which incorporated fibrous CuO particles, demonstrated a more pronounced and dose-dependent antimicrobial effect.⁶⁵ The largest inhibition zone against E. coli was observed for the D5 film (17.4 ± 0.5 mm), followed by D10 (15.0 ± 1.7 mm) and D1 (4.0 ± 1.3 mm). For S. aureus, the inhibition zones were even more prominent, with D10 showing the highest value (19.3 ± 2.7 mm).

Figure 5
Results of the agar disk diffusion assay for PVA films incorporating CuO particles (D, E, and G) at concentrations of 1, 5, and 10%: (a) against E. coli, (b) against S. aureus, both after 24 h of incubation; (c) diameter of the inhibition zone against E. coli, (d) diameter of the inhibition zone against S. aureus

In contrast, the G-series films - containing spherical, well-dispersed CuO particles - did not exhibit detectable inhibition zones, even at the highest particle concentration. This behavior may be attributed to a lower rate of Cu²⁺ ion release or to more effective encapsulation of the particles within the polymer matrix, limiting ion diffusion into the solid medium. Although the direct quantification of Cu²⁺ release was not conducted in this study, previous reports⁶⁶ indicate that morphologies with higher aspect ratios and exposed surface areas tend to promote greater ion availability and stronger interactions with microorganisms in solid environments.

The absence of inhibition halos in G-series films, when contrasted with the results from the CFU assay, suggests that these composites may exhibit superior antimicrobial performance in liquid media, where particle-cell contact is more easily established. This highlights the importance of both the application environment and particle morphology as key factors influencing the antimicrobial efficacy of PVA/CuO films. Lastly, the control films composed of pure PVA did not display any inhibition zones, confirming the lack of inherent antimicrobial activity in the polymer matrix.⁶⁷,⁶⁸

MIC assay

To obtain a more quantitative assessment of bacteriostatic activity, the MIC test was performed using isolated CuO particles. The results revealed that all particle types (D, E, and G) inhibited E. coli growth only at the concentration of 2.5 mg mL^-1, indicating dose dependence. The order of effectiveness was D > G > E, suggesting variations in the antimicrobial potential related to surface structure characteristics and colloidal stability of the particles, which play crucial roles in their interaction with bacterial cells.¹⁶,⁶⁹ According to Karunakaran et al.,⁷⁰ modifications in surface properties, such as functionalization with specific surfactants or polymers, can significantly enhance antibacterial effects depending on the phase and nature of the particles. This suggests that inherent properties of the particles, such as morphology and surface functional groups, play a critical role in their ability to exert inhibitory effects against bacteria.⁷¹

CFU count

The CFU assay revealed distinct results compared to the other tests. As shown in Figure 6, the G series particles led to the greatest reduction in E. coli colony counts, despite being ineffective in the disk diffusion assay. These findings suggest that in liquid media, the G series particles are more effective, possibly due to an increased surface contact area or more efficient release of the active agent. In contrast, the D and E series particles exhibited limited performance under the same conditions.⁷²

Figure 6
CFU assay results for Escherichia coli after treatment with the different CuO particles. Petri dishes show, from left to right: treatments with particles D, E, G, negative control, and positive control, each performed in triplicate

Previous studies support this notion, indicating that the mode of action of antimicrobial agents can vary significantly depending on the physical state of the medium. It has been documented that certain compounds may exhibit strong bactericidal activity in liquid environments due to facilitated interactions with target bacterial cells, emphasizing the dynamic and context-dependent nature of antimicrobial efficacy.⁷³,⁷⁴ In contrast, particles from the D and E series displayed limited effectiveness in liquid media, highlighting variability in response due to chemical composition or physical characteristics.

Moreover, it is well established that CFU reductions greater than three logarithmic scales within a given time frame typically indicate bactericidal activity, whereas smaller reductions may suggest a bacteriostatic effect.⁷³ Thus, while G-series particles significantly reduced E. coli populations in liquid culture, the lower efficacy of D and E particles suggests they may have been more prone to aggregation or less efficient in interacting with bacteria - factors that can be detrimental in liquid environments.⁷⁵

The discrepancies observed between the methods indicate that antimicrobial efficacy depends not only on the presence of the active agent but also on how it interacts with the application environment. The D-series particles, with their fibrous morphology, exhibited greater surface exposure, favoring interaction with microorganisms in direct contact with the film surfaces. In contrast, the G-series particles, which displayed a spherical morphology and were well-dispersed within the matrix, showed better performance in liquid suspension, possibly due to enhanced Cu²⁺ ion release.

Although several studies emphasize advanced strategies such as surface functionalization,⁷⁶ encapsulation, or chemical modification,⁷⁷-⁷⁹ to enhance the interaction between antimicrobial particles and the polymer matrix,⁸⁰ these approaches were not employed in the present study. The CuO particles used here did not undergo any functionalization processes; they were simply physically dispersed in the PVA matrix.

This methodological limitation suggests that the interfacial adhesion between the particles and the matrix may not have been optimized, which could affect the uniformity of dispersion and, consequently, the antimicrobial performance of the films. Nevertheless, the results obtained demonstrated significant effects, indicating that even without functionalization, the incorporation of CuO particles contributes to antimicrobial activity.

CONCLUSIONS

This study demonstrates that the morphology and synthesis route of CuO microparticles play a decisive role in modulating the structural, mechanical, and antimicrobial properties of PVA-based biofilms. The co-precipitation method proved to be effective and reproducible for obtaining particles with distinct morphologies, which in turn influenced particle dispersion, matrix crystallinity, and overall functional performance. XRD analysis, supported by Rietveld refinement, confirmed the formation of highly pure monoclinic CuO phases. SEM observations further revealed that morphological variations arising from synthesis conditions directly affected particle-matrix interactions and distribution uniformity.

The incorporation of CuO particles led to modifications in the crystalline structure of the PVA matrix, with more evident structural reorganization in the G-series films. Mechanically, both fibrous (D-series) and spherical (G-series) particles contributed to enhanced Young’s modulus and tensile strength, with the G5 film standing out for its optimal balance between stiffness and flexibility - attributed to efficient dispersion and interfacial adhesion.

In terms of antimicrobial performance, a morphology-dependent effect was clearly observed. While the D-series films exhibited larger inhibition zones in agar diffusion tests, indicative of effective Cu²⁺ ion release at the film surface, the G-series particles demonstrated superior performance in CFU assays, suggesting more sustained bactericidal activity in liquid media.

However, it is important to highlight that the CuO particles used in this work were not surface-functionalized or chemically modified to enhance compatibility with the polymer matrix. Their incorporation was limited to physical dispersion within the PVA matrix, which may have restricted interfacial adhesion and, consequently, uniformity of dispersion. This methodological limitation suggests that the full potential of these composites may not have been reached.

ACKNOWLEDGMENTS

The authors gratefully acknowledge the CAPES and the Laboratory for Nanomaterials Synthesis and Characterization (LSCN), part of the Brazilian National Nanotechnology Laboratory System (SisNANO), for conducting XRD and FTIR measurements. They also thank the Multiuser Center for the Analysis of Biomedical Phenomena (CMBio) at the University of the State of Amazonas (UEA) for scanning electron microscopy analyses and the Laboratory of Amazonian Materials and Composites (LaMAC) at the Federal University of Amazonas (UFAM) for support with mechanical testing. This work was financially supported by the FAPEAM.

DATA AVAILABILITY STATEMENT

All the data is available in the text.

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Edited by

• Associate Editor handled this article:
  Marcela M. Oliveira

Publication Dates

• Publication in this collection
  20 Apr 2026
• Date of issue
  2026

History

• Received
  06 Aug 2025
• Accepted
  17 Feb 2026
• Published
  10 Mar 2026