A pH-Responsive Polycaprolactone–Copper Peroxide Composite Coating Fabricated via Suspension Flame Spraying for Antimicrobial Applications

Abstract

In this study, a pH-responsive polycaprolactone (PCL)–copper peroxide (CuO₂) composite antibacterial coating was developed by suspension flame spraying. The successful synthesis of CuO₂ nanoparticles and fabrication of the PCL-CuO₂ composite coatings were confirmed by microstructural and chemical analysis. The composite coatings were structurally homogeneous, with the chemical properties of PCL well maintained. The acidic environment was found to effectively accelerate the dissociation of CuO₂, allowing the simultaneous release of Cu²⁺ and H₂O₂. Antimicrobial tests clearly revealed the enhanced antibacterial properties of the PCL-CuO₂ composite coating against both Escherichia coli and Staphylococcus aureus under acidic conditions, with a bactericidal effect of over 99.99%. This study presents a promising approach for constructing pH-responsive antimicrobial coatings for biomedical applications.

1. Introduction

In recent years, bacterial resistance infections have emerged as a significant global health challenge. Biomaterial-associated infections pose a serious threat to global human health [1]. Bacterial resistance to antibiotics can be acquired through mutations in the chromosomal genes or the horizontal transfer of resistance genes, resulting in infections that are difficult to treat [2]. In the medical field, the development of chronic wounds occurs when the healing process of hemostasis, inflammation, hyperplasia, and re-epithelialization is not completed promptly following a skin injury [3,4]. Bacterial infection poses a significant challenge in the management of chronic, non-healing wounds [5]. The interplay between the extended healing time of a wound, which heightens the risk of bacterial infection, and the presence of bacterial infection, particularly drug-resistant strains, which in turn delays wound healing, constitutes a challenging aspect of the management of chronic, hard-to-heal wounds. Therefore, there is a long-standing need for the development of innovative antibacterial materials [6]. These materials may include surface coatings, nanoparticles, or hydrogels designed to overcome the resistance of these microorganisms or enhance the efficacy of antibiotic therapy when used in combination. Metal nanoparticles are currently under investigation for their antimicrobial properties and have shown promise as effective antibacterial agents. Balcucho et al. utilized copper oxide (CuO) metal nanoparticles to fabricate composites capable of releasing Cu²⁺ ions, which showed remarkable growth inhibition of methicillin-resistant Staphylococcus aureus, exceeding four logarithms [7].

Among the various strategies explored, the catalytic treatment of metal peroxide nanoparticles based on the in situ Fenton reaction has garnered considerable attention as a promising antibacterial approach [8,9,10]. The mechanism underlying the Fenton reaction involves the conversion of hydrogen peroxide (H₂O₂) to highly reactive hydroxyl radicals (•OH). It can result in oxidation damage to the membrane and the cell wall and display high and broad-spectrum antibacterial activity compared with traditional antibiotics [11]. Several metal peroxide nanoparticles, such as zinc peroxide (ZnO₂) and calcium peroxide (CaO₂), have been constructed as Fenton reaction–based chemodynamic therapy (CDT) agents [12,13]. Recently, Lin et al. first reported the successful synthesis of CuO₂ nanodots, which could self-supply H₂O₂ in the acidic environment and produce highly toxic •OH via the Fenton reaction between Cu²⁺ and H₂O₂ [14]. CuO₂ is a copper oxide with a unique structure that contains Cu^2⁺ and O₂²⁻ ions in its molecular structure. It has a bent, end-on structure with inequivalent oxygens and peroxide-like O—O distances, typically 1.4–1.55 Å. It maintains the same spin multiplicity as Cu and CuO and presents a controversial ground state in the neutral 3d-metal dioxide series [15]. CuO₂ is synthesized from H₂O₂ and Cu²⁺ under alkaline conditions. Under weak acid conditions, CuO₂ could reversibly decompose into Cu²⁺ and H₂O₂, leading to a Fenton-like reaction between these decomposition products, which in turn generates reactive oxygen species [14]. Both Cu²⁺ and H₂O₂ are well-known antimicrobial agents and have been extensively studied for bacterial infection control. Unlike antibiotics, Cu²⁺ and H₂O₂ are not susceptible to bacterial resistance [16]. CuO₂ demonstrated strong antibacterial effects for biofilm treatment and wound healing [17,18]. The initial environment at the site of bacterial infection is weakly acidic [19], which favors the dissociation of CuO₂ and enables the application of the pH-responsiveness of CuO₂.

However, the inherent instability of CuO₂ under neutral conditions significantly limits its practical application [20]. Additionally, the indiscriminate nature of hydroxyl radicals produced via the Fenton reaction may pose risks of off-target cytotoxicity and tissue damage, highlighting the need for targeted delivery and controlled release strategies to minimize adverse effects. Studies have shown that encapsulation can significantly improve the stability of CuO₂ [18,20]. Compared to other drug delivery systems (such as nanoparticles, electrostatic spinning [21,22], hydrogels [23], gelatin sponges [24,25], etc.), coatings have higher drug-carrying abilities, are easy to store, and can be used to treat large bacterial infections. The thermal spray processes for polymer coating production include flame spraying, high-velocity oxygen fuel (HVOF)/high-velocity air fuel (HVAF), plasma spraying, and cold spraying [26]. Coating biodegradable polymers by flame spraying is a widely used method, with advantages including low cost, simplicity, and environmental friendliness [27].

Polycaprolactone (PCL) is a hydrophobic polyester that has garnered considerable attention in various biomedical applications. This is primarily due to its exceptional biocompatibility, ability to blend with other polymers, distinctive rheological properties, and controlled release of active compounds. These characteristics are closely tied to its biodegradability [7,28]. Therefore, PCL presents itself as a promising choice for integration as a structural component of dressings that come into direct contact with living tissue. The incorporation of CuO₂ nanoparticles into a PCL matrix not only preserves the intrinsic properties of the nanoparticles but also extends their stability and facilitates their controlled release [29].

This study focuses on developing a biodegradable and biocompatible material for antimicrobial applications in the weakly acidic microenvironment. An innovative approach using the suspension flame spraying method was employed to fabricate pH-responsive antimicrobial coatings with different contents of CuO₂ nanoparticles in the PCL matrix. Various analyses were conducted to confirm the successful incorporation of CuO₂. Their release mechanisms and antimicrobial properties were also examined under different pH conditions. The study offers a universal fabrication method for pH-responsive CuO₂-loaded composite coatings for effectively combating biomaterial-associated infections.

2. Materials and Methods

2.1. Materials and Reagents

Copper (II) chloride dihydrate (CuCl₂·2H₂O), polyvinylpyrrolidone (PVP, MW of ~10,000), hydrogen peroxide (H₂O₂, 30%), sodium hydroxide (NaOH), sulfuric acid (H₂SO₄), and potassium permanganate (KMnO₄) were purchased from Sinopharm Group Co., Ltd., Shanghai, China. PCL powders (200 mesh, MW of ~80,000) were provided by Nature Works, Minneapolis, MN, USA.

2.2. Sample Preparation

CuO₂ was synthesized according to Lin et al. [14] with slight modifications. To begin, 5 g PVP was added into 50 mL of aqueous solution containing 0.05 M CuCl₂. After that, 50 mL of 0.10 M NaOH and 5 mL of 30% H₂O₂ were sequentially incorporated into the above mixture solution. After stirring for 30 min, the resulting nanoparticles were separated and purified to obtain the CuO₂ powder after freeze-drying.

PCL powders were suspended in 50:50 (v/v) ethanol/water, and then 0.1%, 0.3%, and 0.6% (wt/wt) CuO₂ powders relative to PCL powders were added to prepare the PCL-CuO₂ coatings. The coatings were prepared by flame spraying (CDS 8000, Castolin, Kriftel, Germany) on 316 L stainless-steel plates with dimensions of 25 × 20 × 2.0 mm [30]. The suspension was injected into the flame using a homemade spray atomizer. Acetylene was used as the fuel gas, with a flow rate of 1.5 Nm³/h and a working pressure of 0.1 MPa. Oxygen was used as the combustion gas, with a flow rate of 2.5 Nm³/h and a working pressure of 0.5 MPa. The spray distance was 200 mm. The thicknesses of PCL and PCL-CuO₂ coatings were about 320–380 μm, measured by the coating thickness gauge. A schematic diagram of the preparation of the coating by liquid flame spraying is shown in Figure 1.

2.3. Sample Characterization

The microstructures of the powders and coatings were examined using a field emission scanning electron microscope (SEM, S4800, Hitachi, Tokyo, Japan). The cross sections of the coatings were characterized using an energy dispersive X-ray detector (EDX, XFlash 6–100, Bruker, Ettlingen, Germany). The particle size and zeta potential of CuO₂ were measured by nanometer particle size analyzer (Litesizer 500, Anton Paar, Graz, Austria). Chemical composition was characterized using X-ray photoelectron spectroscopy (XPS, AXIS ULTRA DLD, Shimadzu, Kyoto, Japan). The X-ray diffraction (XRD) patterns were obtained on a D8-Advance X-ray diffractometer (Bruker, Germany), with Cu Kα radiation at a voltage of 40 kV and a tube current of 40 mA. Chemistry of the powders and coatings was detected by Fourier transform infrared spectroscopy (FT-IR, Nicolet iS50, Thermo Scientific, Waltham, MA, USA), operated at a spectral resolution of 4 cm⁻¹ with a scan range of 4000~400 cm⁻¹.

2.4. Colorimetric Determination of Peroxo Groups

KMnO₄ solution is a strong purplish-red oxidizer that can oxidize H₂O₂, thus causing the purplish-red color to fade [14]. KMnO₄ was dissolved in 0.1 M aqueous H₂SO₄ solution to obtain a concentration of 50 μg/mL, and then the acidic KMnO₄ solution was treated with H₂O (control), H₂O₂, Cu(OH)₂, freshly synthesized CuO₂, and the CuO₂ suspension placed at room temperature for a short period, consecutively. After 10 min of incubation, photographs were taken to record the fading, and UV-vis spectra were examined at 400–650 nm.

2.5. pH-Responsive Release of Copper Ions

The areas surrounding the coating and the back of the substrate were sealed with epoxy resin, ensuring that only the coating surface remained exposed. For the release analysis, three parallel samples were used for each group of PCL-CuO₂ composite coatings and each pH condition. Generally, coating samples were individually immersed in 15 mL PBS buffer at pH 7.4 or pH 5.5 in 50-mL falcon tubes with gentle shaking at 37 °C. At predetermined time intervals, 6 mL solution was collected with the addition of 6 mL fresh PBS. The content of copper ions was determined using inductively coupled plasma optical emission spectroscopy (ICP-OES; SPECTRO ARCOS, SPECTRO Analytical Instruments, Kleve, Germany).

2.6. In Vitro Antibacterial Effect of PCL-CuO₂ Coatings

Escherichia coli (E. coli, ATCC25922) and Staphylococcus aureus (S. aureus, ATCC6538) were used to evaluate the antimicrobial effect of the coatings. We performed the tests according to the Japanese Industrial Standard (JIS) Z 2801 (ISO 22196:2011 [31], measurement of antibacterial activity on plastics and other nonporous surfaces) with some modifications [7]. Three parallel samples of each group of coatings were placed in sterile 6-well plates, and the bacteria cultured to logarithmic growth phase were diluted to about 1×10⁶ CFU/mL by gradient dilution in PBS at pH 7.4 or pH 5.5. Then, 10 μL bacterial suspension was dropped on the coating surface and covered with a sterile polyethylene film (5 × 5 mm) and then incubated at 37 °C with a relative humidity of at least 95% for 2 h. Afterward, bacteria were collected by washing with 1 mL PBS buffer and used for determination of survival rate by standard plate counting method. LB medium was used to grow E. coli, and TSB was used to grow S. aureus. The bactericidal effect can be calculated using the formula: R = (N₀ − N₁)/N₀ × 100%, where N₀ represents the number of bacterial colonies in the control group and N₁ represents the number of bacterial colonies in the experimental group.

3. Results and Discussion

3.1. Morphological Characterization of PCL and CuO₂ Powders

The morphology of commercially available PCL powders and synthesized nanosized CuO₂ particles was examined by SEM analysis. The PCL powders showed irregular morphology and a wide size range, roughly from 10 to 60 μm (Figure 2a). The synthesized CuO₂ particles were formed by aggregation of low nanosized particles and showed irregular shape and good dispersion (Figure 2b).

3.2. The Particle size and Zeta Potential of CuO₂

The particle size analysis showed that the average hydrodynamic diameter of the CuO₂ was 163 ± 1.80 nm (Figure 3a). The results are consistent with the SEM results described above. Zeta potential is a good indicator of the magnitude of electrostatic interactions between dispersed particles and can be used as a reference for the stability of nanoparticle dispersions [32]. The average zeta potential carried by CuO₂ was 20.6 ± 2.9 mV (Figure 3b), indicating that it has good dispersibility.

3.3. XPS Analysis of CuO₂ Powders

The X-ray photoelectron (XPS) spectrum of the fully scanned region of CuO₂ powders exhibited characteristic peaks of C 1s, N 1s, O 1s, and Cu 2p (Figure 4a). The peaks of C 1s and N 1s indicated the presence of PVP. The XPS spectrum of Cu 2p displayed characteristic peaks at 953.9 eV and 933.6 eV, respectively, accompanied by two satellite peaks at 962.1 eV and 942.1 eV, respectively, indicating that the valence state of copper in CuO₂ is +2 [14] (Figure 4b). Furthermore, the O 1s XPS spectrum showed three distinct peaks at 529.5, 531.5, and 533.0 eV, ascribed to Cu-O, C=O, and O-O bonds, respectively [33]. The presence of peroxo groups in the synthesized CuO₂ powder was confirmed by the presence of the O-O bond (Figure 4c). The XPS spectrum of C 1s showed three characteristic peaks at 284.8, 286.3, and 288.3 eV, which were assigned to C-C, C-N, and C=O, respectively [34] (Figure 4d). The above indicated the successful preparation of CuO₂ nanoparticles.

3.4. Potassium Permanganate Colorimetric Analysis of Synthesized CuO₂

Furthermore, a KMnO₄-based colorimetric method was used to examine the synthesized CuO₂ powders. The absorption peaks of MnO₄⁻ disappeared when mixed with H₂O₂ or the synthesized CuO₂ powder but remained when mixed with H₂O or Cu(OH)₂ (Figure 5). It also suggests the presence of peroxo groups in the synthesized CuO₂ powders, which is consistent with the above XPS results. However, CuO₂ in water is unstable and easily decomposed [20]. As shown in Figure 5, the CuO₂ suspension almost completely lost the ability to decolorize potassium permanganate after sitting at room temperature for 7 days, indicating the fast decomposition of CuO₂.

3.5. SEM and EDX Analysis of the PCL and PCL-CuO₂ Coatings

Figure 6a–d demonstrated that PCL and PCL-CuO₂ coatings had smooth surfaces with visible pores. This might result from the fast evaporation of deionized water or ethanol during the manufacturing process [35]. There were no visible CuO₂ particles on the surface or cross-section, which might be due to the homogeneous entrapment of PCL [36], as shown in Figure 6(a–d,a-1–d-1). These results demonstrated that the addition of CuO₂ has no significant impact on either the surface or internal structure of the PCL coating. Furthermore, an enrichment of Cu was shown in the PCL-0.6% CuO₂ coating in Figure 7, indicating the incorporation of CuO₂ nanoparticles within the PCL matrix.

3.6. XRD Analysis of the Powders and Coatings

In Figure 8, the XRD pattern analysis showed that the PCL powder exhibited intense diffraction peaks at 21.8°, 22.5°, and 24.2°, which are assigned to the planes (110), (111), and (200) of PCL, respectively [37]. The XRD pattern of the synthesized CuO₂ nanoparticles was consistent with that reported in the literature, with two envelope peaks at 32.3° and 38.8° [33], indicating poor crystallization of the synthesized CuO₂ [38]. These two envelope peaks were not observed in the PCL-CuO₂ composite coating, which might be due to the low content of CuO₂. These results indicated the successful synthesis of CuO₂ nanoparticles and fabrication of PCL-CuO₂ coatings.

3.7. FT-IR Analysis of the Powders and Coatings

The FT-IR spectra of PCL powder, CuO₂ powder, PCL coating, and PCL-CuO₂ composite coatings were analyzed, as shown in Figure 9. For PCL powder, the peak at 2956 cm⁻¹ was the asymmetric stretching vibration of CH₂, and the characteristic peak at 1727 cm⁻¹ was the stretching vibration of the C=O group. The peak of stretching vibration of C-H at 1464 cm⁻¹, 1370 cm⁻¹ belongs to CH₂, and the peak of asymmetric stretching vibration of C-O-C group at 1259 cm⁻¹, 1165 cm⁻¹ belongs to C-O. The peaks at 1067 cm⁻¹, 960 cm⁻¹, and 732 cm⁻¹ represented the C-C, C-O-C, and CH₂ vibration peaks, respectively [39]. There was no significant difference in the position of the infrared absorption peak between the PCL powder and the prepared coating, indicating that the chemical composition of the PCL coatings prepared by suspension flame spraying was not changed. The characteristic peaks at 1652 cm⁻¹ and 1290 cm⁻¹ in the CuO₂ powder are attributed to the tensile vibration between C=O and C-N in PVP [40], and the two small peaks displayed at 1464 cm⁻¹ and 1370 cm⁻¹ are the characteristic peaks of peroxide group (O-O) [41]. The above characteristic peaks of the CuO₂ powder were not detected in the PCL-CuO₂ coatings, which might be due to the low content of CuO₂ in the composite coatings.

3.8. pH-Responsive Release of PCL-CuO₂ Coatings

Under weakly acidic conditions, CuO₂ decomposes to produce Cu²⁺ and H₂O₂, which further produces reactive oxygen species via the Fenton reaction [14]. The release of Cu²⁺ from the PCL-CuO₂ coatings was examined under different pH conditions (Figure 10). As expected, increasing Cu²⁺ release with the content of CuO₂ was clearly observed at pH 5.5, while the release at pH 7.4 was negligible. At pH 5.5, the copper ion release from PCL-0.1% CuO₂ coating for 7 days was 0.15 mg/L; from PCL-0.3% CuO₂ coating, it was 0.41 mg/L; and from PCL-0.6% CuO₂ coating, it was 1.45 mg/L. However, the release of copper ions from PCL-0.1% CuO₂, PCL-0.3% CuO₂, and PCL-0.6% CuO₂ coatings at pH 7.4 for 7 days were only 0.01, 0.02, and 0.04 mg/L, respectively. The above indicated that the PCL-CuO₂ coatings showed sustained release in an acid-responsive and dose-dependent manner. A previous study reported that a concentration of Cu²⁺ of no more than 9 ppm showed no significant effect on the growth of cells compared with normal conditions [42]. In this study, the release of Cu²⁺ was very slow, and the concentration of Cu²⁺ after 7 days continuous release was significantly lower than 9 ppm, suggesting high biocompatibility of the PCL-CuO₂ coatings.

Furthermore, the data were fitted with four commonly used drug release models (Figure 11a–d) and listed in

Table 1. The variables calculated from the release kinetics of Cu^2+.

Sample		7.4 0.1%	7.4 0.3%	7.4 0.6%	5.5 0.1%	5.5 0.3%	5.5 0.6%
Zero-order model	K0 (×10−5)	0.62	0.24	0.42	8.84	7.94	15.55
	R2	0.691	0.449	0.971	0.902	0.774	0.953
First-order model	K1 (×10−5)	−0.62	−0.24	−0.42	−8.92	−8.01	−15.78
	R2	0.691	0.449	0.971	0.904	0.776	0.955
Higuchi model	KHI (×10−5)	0.90	0.35	0.55	12.5	11.8	21.5
	R2	0.786	0.521	0.944	0.984	0.943	0.995
Korsmeyer–Peppas model	KKP (×10−5)	0.08	3.71	0.75	20.29	18.36	12.31
	R2	0.879	0.108	0.764	0.957	0.964	0.997
	n	0.99	0.06	0.39	0.40	0.43	0.61

. It was noted that, under pH 5.5, the release for the PCL-0.3% CuO₂ and PCL-0.6% CuO₂ coatings fitted the Korsmeyer–Peppas model, with the highest linearity correlation coefficient (R² = 0.964, 0.997). The release exponent n for PCL-0.3% CuO₂ was ≤0.45, indicating the drug release mechanism follows Fick’s laws of diffusion. On the contrary, the release exponent n for PCL-0.6% CuO₂ was higher than 0.45, suggesting a combined erosion and diffusion release mechanism, named non-Fickian transport. The release of the PCL-0.1% CuO₂ coating was best interpreted by the Higuchi equation (R² = 0.984), indicating a relatively slower diffusion from the PCL matrix. However, it was difficult to define the release models of the PCL-CuO₂ coatings under pH 7.4, which generally showed an R² value lower than those under pH 5.5. This might result from the extremely slower release of the composite coatings under pH 7.4. The complexity of the release mechanisms for these coatings might be due to the combined impact of CuO₂ diffusion from the coating and the decomposition reaction of CuO₂.

3.9. In Vitro Antibacterial Properties of PCL-CuO₂ Coatings

The decomposition of CuO₂ displays a pH-responsive manner and can continuously release copper ions and H₂O₂ under weakly acidic conditions. Therefore, the antibacterial effect of the PCL-CuO₂ composite coating was examined at pH 5.5 and 7.4 using E. coli and S. aureus. It was found that the composite coatings exhibited a significantly higher antibacterial effect against both E. coli and S. aureus at pH 5.5 compared to pH 7.4 (Figure 12a,b). Furthermore, the antibacterial effect of the composite coatings was dose-dependent and reached over 99.99% killing efficacy against both E. coli and S. aureus for 0.6% CuO₂ content at pH 5.5. Moreover, the composite coatings displayed superior killing efficacy for S. aureus compared to E. coli under both pH conditions, which might be due to there being a difference in the antimicrobial activity of metal nanoparticles depending on the bacterial wall structure [7]. Gram-positive bacteria with higher peptidoglycan and cell wall protein content are more sensitive to copper [16,43]. Excitingly, there is no significant change in the antimicrobial effect of these coatings after 10 months (Figure S1), suggesting the excellent stability of CuO₂ within the PCL-CuO₂ coatings.

4. Conclusions

In this research, PCL-CuO₂ composite coatings with pH-responsive antimicrobial properties were fabricated using a suspension flame spraying technique. Morphology and chemical characterization confirmed the well-maintained PCL matrix and the successful incorporation of CuO₂ nanoparticles in the composite coatings. The release study found that the PCL-CuO₂ coatings showed sustained release in an acid-responsive and dose-dependent manner. The composite coating with 0.6% (w/w) CuO₂ exhibited over 99.99% antibacterial effect against E. coli and S. aureus under a mildly acidic (pH 5.5) condition. The CuO₂ nanoparticles in the PCL-CuO₂ composite coatings showed significantly enhanced stability in comparison with fast decomposition in an aqueous solution and still exhibited potent antimicrobial efficacy after 10 months storage. Our cost-effective fabrication method of pH-responsive antimicrobial coatings provides a new solution for the development of biomedical materials for various applications.