Boosting the Anti-Infection Ability of Titanium Implants by Coating Polydopamine–Curcumin

: To reduce the risk of infection, improving the anti-infection ability of Ti-based implantation has become a very meaningful task. In this work, by employing polydopamine (PDA) as a carrier and curcumin (CUR) as an anti-biotic/inflammatory, a series of Ti-PDA@CURx (x = 0.5, 1.0, 1.5, 2.0, 2.5) was successfully fabricated and characterized by scanning electron microscopy, Fourier transform infrared spectrometry, X-ray diffraction, X-ray photoelectron spectroscopy, and the water contact angle, where Ti plates were firmly coated by PDA@CUR. The test result of CUR content shows that the maximum loading of CUR in PDA can reach 0.6506%, where the CUR concentration is 1.5 mg/mL. The antibacterial test results demonstrate that Ti-PDA@CUR-x (x = 0.5, 1.0, 1.5, 2.0, 2.5) exhibit significant antibacterial activities against Escherichia coli and S. aureu , where Ti-PDA@CUR1.5 has the highest antibacterial rate of 62.7% against Escherichia coli and 52.6% against S. aureus . The cytotoxicity test shows that Ti-PDA, Ti-PDA@CUR-x (x = 0.5, 1.0, 1.5, 2.0, 2.5) has almost no toxicity.


Introduction
Because of their excellent mechanical properties, corrosion resistance, and biocompatibility, Ti-based implants have received increasing attention in dentistry, bone integration, and joint replacement surgery [1][2][3][4].However, it was found that approximately 5% of patients receiving treatment were infected with bacteria after surgery because Ti-based implants do not have antibacterial properties [5][6][7][8].To reduce the risk of implant infection, antibiotics are frequently introduced into Ti-based implantation [9], but just like the two sides of a coin, the overuse of antibiotics can result in the emergence of antibiotic-resistant bacteria in patients [10][11][12][13].Once bacterial infection occurs, it can lead to implant failure, which requires surgical removal, resulting in surgical failure and economic losses [14].Therefore, many antibacterial agents have been loaded on a Ti substrate to fabricate an antibacterial coating, and thereby, the antibacterial ability of Ti-based implants has been greatly improved, including metal [15,16] compounds/metal ions/nanoparticles [17] and antibiotics [18].However, the use of metal layers as antibacterial agents may generate many side effects on the body's immunity and metabolism [19,20].Therefore, developing a new coating for Ti-based implants with antibacterial properties and no cytotoxicity is very necessary.
Curcumin (CUR), a component abstracted from turmeric, possesses antibacterial [21,22], anti-inflammatory [23], and anti-tumor effects [24,25].It is cost-effective, safe, and efficient, with minimal side effects, making it particularly well-suited for use in antibacterial applications [26].However, curcumin is difficult to load tightly and heavily with Ti-based implants, as curcumin molecules contain multiple double bonds, as well as active groups such as phenolic hydroxyl and carbonyl groups, which can chemically bind with most compounds [27].Its structure is shown in Figure 1 (right).It is well known that dopamine rial applications [26].However, curcumin is difficult to load tightly and heavily with Tibased implants, as curcumin molecules contain multiple double bonds, as well as active groups such as phenolic hydroxyl and carbonyl groups, which can chemically bind with most compounds [27].Its structure is shown in Figure 1 (right).It is well known that dopamine can undergo oxidation and self-polymerization to create a polydopamine (PDA) coating that covers nearly every solid surface under alkaline conditions.In 2012, PDA was first discovered to be essential for the adhesion of sessile mussels [28] because catechol and primary amine, the functional groups of PDA, have better adhesion and antioxidant properties compared with others [29,30].Its structure is shown in Figure 1 (left).Moreover, PDA is a commonly used drug carrier with good stability, hydrophilicity, and biocompatibility [31,32].Considering the facts mentioned above, herein, by employing PDA as a carrier and CUR as an anti-biotic/inflammatory, a series of Ti-PDA@CUR was designed and fabricated, and the CUR loading contents (Ti-PDA@CUR-x (x = 0.5, 1.0, 1.5, 2.0, 2.5) were explored.More importantly, Ti-PDA@CURx exhibit excellent antibacterial activities against Escherichia coli and S. aureu and no toxicity.This work offers a promising research direction for the clinical application of antibacterial Ti-based implants.
This study employs a simple and effective method for modifying CUR on the surface of titanium metal in order to explore the antibacterial effect of a CUR coating on Escherichia coli and Staphylococcus aureus, as well as the cytotoxic effect on osteoblast 3T3-E1.

Materials and Specimen Fabrication
Pure titanium plates for commercial use (Cp Ti; TA1, purity > 99.85%) measuring 10 × 10 × 1 mm 3 were utilized.Each Ti plate was ultrasonically cleaned with a cleaning solution (V :V :V = 1:5:34).Initially, 1.21 g Tris (China Pharmaceutical Group, Shanghai, China) was weighed and placed in a 1 L beaker.Approximately 800 mL of deionized water was added to the beaker and stirred thoroughly to dissolve.The pH was adjusted to 8.5 using 1% hydrochloric acid, and the solution was brought to 1 L followed by the preparation of a 2 mg/mL dopamine solution by dissolving dopamine hydrochloride (McLean Reagent Co., Ltd., Shanghai, China) in a Tris-HCl buffer solution.Afterward, the titanium plate was immersed in a dopamine solution for 24 h.The soaking process was carried out on a constant temperature shaker at a constant speed, avoiding light vibrations.Next, the sample was rinsed with DI water to produce Ti-PDA after 24 h.Finally, Ti-PDA, as the substrate, was immersed in a CUR solution (0.5 mg/mL, 1.0 mg/mL, 1.5 mg/mL, 2.0 mg/mL and 2.5 mg/mL) for 24 h following the same preparation procedures and resulting in Ti-PDA@CUR0.5,Ti-PDA@CUR1.0,Ti-PDA@CUR1.5,Ti-PDA@CUR2.0,and Ti-PDA@CUR2.5.

Test of the CUR Content
The samples of Ti-PDA@CUR-x (x = 0.5, 1.0, 1.5, 2.0, 2.5) were sonicated in an ethanol solution to obtain five different concentrations of the CUR solution.The Ti sheets were placed in anhydrous ethanol to accelerate its release under ultrasound conditions.During this process, the solution was changed several times until it was released under ultrasound This study employs a simple and effective method for modifying CUR on the surface of titanium metal in order to explore the antibacterial effect of a CUR coating on Escherichia coli and Staphylococcus aureus, as well as the cytotoxic effect on osteoblast 3T3-E1.

Materials and Specimen Fabrication
Pure titanium plates for commercial use (Cp Ti; TA1, purity > 99.85%) measuring 10 × 10 × 1 mm 3 were utilized.Each Ti plate was ultrasonically cleaned with a cleaning solution (V HF :V HNO 3 :V H 2 O = 1:5:34).Initially, 1.21 g Tris (China Pharmaceutical Group, Shanghai, China) was weighed and placed in a 1 L beaker.Approximately 800 mL of deionized water was added to the beaker and stirred thoroughly to dissolve.The pH was adjusted to 8.5 using 1% hydrochloric acid, and the solution was brought to 1 L followed by the preparation of a 2 mg/mL dopamine solution by dissolving dopamine hydrochloride (McLean Reagent Co., Ltd., Shanghai, China) in a Tris-HCl buffer solution.Afterward, the titanium plate was immersed in a dopamine solution for 24 h.The soaking process was carried out on a constant temperature shaker at a constant speed, avoiding light vibrations.Next, the sample was rinsed with DI water to produce Ti-PDA after 24 h.Finally, Ti-PDA, as the substrate, was immersed in a CUR solution (0.5 mg/mL, 1.0 mg/mL, 1.5 mg/mL, 2.0 mg/mL and 2.5 mg/mL) for 24 h following the same preparation procedures and resulting in Ti-PDA@CUR0.5,Ti-PDA@CUR1.0,Ti-PDA@CUR1.5,Ti-PDA@CUR2.0,and Ti-PDA@CUR2.5.

Test of the CUR Content
The samples of Ti-PDA@CUR-x (x = 0.5, 1.0, 1.5, 2.0, 2.5) were sonicated in an ethanol solution to obtain five different concentrations of the CUR solution.The Ti sheets were placed in anhydrous ethanol to accelerate its release under ultrasound conditions.During this process, the solution was changed several times until it was released under ultrasound conditions, the Ti-PDA@CURx (x = 0.5, 1.0, 1.5, 2.0, 2.5) of the solution was clear and transparent, and there was no absorbance of the solution under a UV spectrophotometer.The absorbance was measured at 426 nm using a UV visible spectrophotometer (UV-5200 PC, Shanghai, China), and the CUR loading amount in the sample was determined by substituting the absorbance into the CUR standard curve.The calculation formula is as follows: Coatings 2024, 14, 640 3 of 11 CUR loading efficiency (%) = Weight of the loaded CUR Weight of the initial sample × 100%

In Vitro Antibacterial Tests 2.4.1. Bacterial Culture
Bacterial solutions of E. coli (ATCC 25922) and S. aureus (ATCC 25923) were streaked and inoculated on the surface of a nutrient agar plate with a 200 µL pipette tip, which was cultured in a bacterial culture incubator for 15 h.After passing to the third generation without impurities, they were used as experimental strains.The bacterial strains were removed and inoculated into nutrient an agar medium (TSB (McLean Biochemical Technology Co., Ltd., Shanghai, China) for Gram-positive bacteria and LB (McLean Biochemical Technology Co., Ltd., Shanghai, China) for Gram-negative bacteria) for cultivation for 5 h.

Bacteriostatic Rate Test
First, 60 µL of E. coli and S. aureus were inoculated at a concentration of 1 × 10 7 CFU/mL on the antimicrobial susceptibility tablets prepared as described above and grown in an incubator at 37 • C.After 24 h, the microbes present on the surface of the susceptibility tablets were rinsed with saline solution and then thinned to a concentration of 1 × 10 6 CFU/mL to create a bacterial suspension.Then, 100 µL of the diluted bacterial solution was inoculated on the agar plates, and the bacterial solution was spread evenly on the agar plates with the applicator.After incubation in a 37 • C incubator for 18 h, the agar plates with colonies were taken, and the number of colonies was measured using Image J 1.8.0 software.
Bacteriost atic rate (% where A represents the number of colonies recovered from the Ti-PDA sample and B represents the number of colonies recovered from the test samples.

Zone of Inhibition
The resulting susceptibility tablets were pasted on the surface of E. coli and S. aureus agar plates.Plates with susceptibility tablets were incubated in a 37

Bacterial Crystal Violet Staining
The antimicrobial properties of the susceptibility tablets were further assessed by bacterial biofilm crystal violet staining [35].After preparing the groups of susceptibility tablets as described above, they were placed in 24-well plates.E. coli and S. aureus at a concentration of 1 × 10 6 CFU/mL were inoculated onto the tablet surfaces.The plates were incubated at 37 • C for 48 h before the bacteria were removed.Subsequently, the plates were washed thrice with PBS (McLean Reagent Co., Ltd., Shanghai, China, AR > 98%) to eliminate any inactive bacteria.Finally, 2.5% glutaraldehyde (McLean Reagent Co., Ltd., Shanghai, China) was added, and the samples were fixed at 4 • C for 4 h.After eliminating glutaraldehyde, 0.5% crystal violet dye was applied to every well plate for a duration of 2 h, and the residual dye solution was removed by washing the well plates three times with PBS.Pictures were taken to record the crystal violet staining in the different groups.

Cytotoxicity Test
DMEM medium (Bioengineering Co., Ltd., Shanghai, China) was set as a blank control group, and Ti-PDA, Ti-PDA@CUR0.5,Ti-PDA@CUR1, Ti-PDA@CUR1.5,Ti-PDA@CUR2, and Ti-PDA@CUR2.5 were set as the experimental groups.Each group of samples (three parallel samples per group) was placed on a super clean workbench, irradiated with ultraviolet light at a wavelength of 260 nm for 12 h, and then used later.
Experiments were performed on 3T3-E1 cells in the logarithmic growth phase, which were washed twice with PBS.First, 1 mL of trypsin cell digestion solution (0.25% trypsin) was added for 1 min, then 1 mL of DMEM medium was added to terminate digestion.The cell suspension was blown to form and centrifuged at 1000 rpm/min for 5 min.The supernatant was removed, and 1 mL of DMEM medium was added, blown to form cell suspension, and counted.The samples were placed in a 24-well plate in the order of grouping.The cells were inoculated into well plates at a density of 5 × 10 4 per well, and 500 µL cells per well were contained in DMEM complete culture medium after 48 h of cultivation in the incubator.The samples were removed and 20 µL of CCK-8 liquid and 180 µL of DMEM basic culture medium were added to each well, according to the instructions of the CCK-8 reagent kit, and gently shaken to make it uniform.Next, the cells were further grown in the incubator for 2 h.Subsequently, each group of solutions was moved to a 96-well plate, and the optical density (OD) of each sample in the wells was assessed using a microplate reader (BioTek-Infinire M200pro, Tecan, Männedorf, Switzerland) at a wavelength of 450 nm.The relative proliferation rate (RGR%) of 3T3-E1 cells was calculated.The calculation formula is as follows: RGR% = ODe ODc × 100% where ODe represents the mean OD value of the experimental group, while ODc represents the mean OD value of the control group.According to the standards of the U.S.
Pharmacopoeia, the cytotoxicity level (CTG) was evaluated, as shown in Table 1.

Statistical Analysis
The experimental data were analyzed statistically using SPSS (IBM SPSS Statistics 19) software, and the results were reported as the mean ± standard deviation.

Drug Loading Efficiency
Figures 2a and 2c, respectively, show the UV absorption spectra of CUR at 426 nm and the standard curves of CUR.In Figure 2b, as the concentration of CUR preparation solution increases, the loading of CUR in Ti-PDA significantly increases; when the CUR concentration is 1.5 mg/mL, the maximum loading of CUR in Ti-PDA reaches 0.6506%.When the CUR concentration exceeds 1.5 mg/mL, the loading of CUR in Ti-PDA shows a slow decreasing trend and finally tends to equilibrium at 2.5 mg/mL.Possible explanations are as follows: the catechol groups of CUR, and the primary amino groups of PDA can interact through hydrogen bonding [36], and the interaction force between CUR and PDA reaches the maximum or saturation when the CUR concentration is 1.5 mg/mL.As the concentration of CUR continues to increase, the repulsive force increases, hydrogen bonds weaken, and the loading decreases [37].
Coatings 2024, 14, x FOR PEER REVIEW 5 of 12 The experimental data were analyzed statistically using SPSS (IBM SPSS Statistics 19) software, and the results were reported as the mean ± standard deviation.

Drug Loading Efficiency
Figure 2a and Figure 2c, respectively, show the UV absorption spectra of CUR at 426 nm and the standard curves of CUR.In Figure 2b, as the concentration of CUR preparation solution increases, the loading of CUR in Ti-PDA significantly increases; when the CUR concentration is 1.5 mg/mL, the maximum loading of CUR in Ti-PDA reaches 0.6506%.When the CUR concentration exceeds 1.5 mg/mL, the loading of CUR in Ti-PDA shows a slow decreasing trend and finally tends to equilibrium at 2.5 mg/mL.Possible explanations are as follows: the catechol groups of CUR, and the primary amino groups of PDA can interact through hydrogen bonding [36], and the interaction force between CUR and PDA reaches the maximum or saturation when the CUR concentration is 1.5 mg/mL.As the concentration of CUR continues to increase, the repulsive force increases, hydrogen bonds weaken, and the loading decreases [37].

Characterization of Specimens
SEM images of Ti, Ti-PDA, and Ti-PDA@CUR1.5 are shown in Figure 3a-c.Compared with Ti, the surface of Ti-PDA is covered with a uniform layer of spherical nanoparticle coating.After loading CUR, block-shaped CUR can be clearly observed [38].The FT-IR spectra of Ti-PDA, CUR, and Ti-PDA@CUR1.5 were also explored.As shown in Figure 3d, after CUR loading, new absorption peaks appear at 1607 cm −l and 1508 cm −1 in Ti-PDA@CUR1.5, which are attributed to the vibrations of -C=0 and -C=C in CUR.In addition, the peaks at 1000-1300 cm −1 can be assigned to the stretching vibrations of C-O-C in CUR [39].Meanwhile, the peaks between 1000 and 1300 cm −1 may belong not only to C-O-C but also to C-O, C-C, and other functional groups.The peaks at about 900 and 650 cm-1 indicate interactions between Ti and oxygen (Ti-O) [40].Furthermore, the peaks in the range of 3100-3500 cm −1 in Ti-PDA@CUR1.5 are more prominent than those of CUR, indicating the presence of strong bands related to the stretching vibration of -NH and -OH within PDA, unlike CUR [41].As shown in Figure 3e, peaks at 18.98° and 27.81° are attributed to CUR and those at 12.13° originate from Ti-PDA [42], which can be observed in the XRD of Ti-PDA@CUR1.5, indicating crystalline CUR attached well to the Ti-PDA surface [43,44].Based on the above experimental results, it is shown that the catechol group of CUR and the primary amino group of PDA successfully bind together under hydrogen bonding [36], proving that the Ti-PDA@CUR coating was successfully prepared.

Characterization of Specimens
SEM images of Ti, Ti-PDA, and Ti-PDA@CUR1.5 are shown in Figure 3a-c.Compared with Ti, the surface of Ti-PDA is covered with a uniform layer of spherical nanoparticle coating.After loading CUR, block-shaped CUR can be clearly observed [38].The FT-IR spectra of Ti-PDA, CUR, and Ti-PDA@CUR1.5 were also explored.As shown in Figure 3d, after CUR loading, new absorption peaks appear at 1607 cm −l and 1508 cm −1 in Ti-PDA@CUR1.5, which are attributed to the vibrations of -C=O and -C=C in CUR.In addition, the peaks at 1000-1300 cm −1 can be assigned to the stretching vibrations of C-O-C in CUR [39].Meanwhile, the peaks between 1000 and 1300 cm −1 may belong not only to C-O-C but also to C-O, C-C, and other functional groups.The peaks at about 900 and 650 cm −1 indicate interactions between Ti and oxygen (Ti-O) [40].Furthermore, the peaks in the range of 3100-3500 cm −1 in Ti-PDA@CUR1.5 are more prominent than those of CUR, indicating the presence of strong bands related to the stretching vibration of -NH and -OH within PDA, unlike CUR [41].As shown in Figure 3e, peaks at 18.98 • and 27.81 • are attributed to CUR and those at 12.13 • originate from Ti-PDA [42], which can be observed in the XRD of Ti-PDA@CUR1.5, indicating crystalline CUR attached well to the Ti-PDA surface [43,44].Based on the above experimental results, it is shown that the catechol group of CUR and the primary amino group of PDA successfully bind together under hydrogen bonding [36], proving that the Ti-PDA@CUR coating was successfully prepared.
Finally, the chemical composition of CUR, Ti-PDA, and Ti-PDA@CUR1.5 was investigated by XPS, and the survey spectra prove the coexistence of Ti, C, N, and O [34,41] (Figure 4a).The high-resolution spectra of C 1s could be fit with three peaks at ca. 284.8 eV and 286.1 eV, corresponding to C-C and C-O, respectively (Figure 4b).The deconvoluted peaks at ca. 400.0 eV, 401.6 eV, and 402.8 eV can be observed in the spectrum of N 1s (Figure 4c), which are attributed to -NH=, -NH-, and -N + H-, respectively, where the -NH-bond serves as the connection between PDA and CUR [37].Additionally, C=O and C-O bonds can be detected at 531.3 eV and 533.1 eV in the spectrum of O 1s (Figure 4d).According to Yan's calculation method [33], compared with Ti-PDA, after coating CUR, the content of C-C bonds (59.4-28.1 eV) and C=O bonds (9.1-6.9 eV) decreased, and the content of -NH-bonds increased (31.8-67.2eV) in Ti-PDA@CUR1.5,further indicating that the Ti-PDA@CUR composite was successful and there was a strong interaction force between them.Finally, the water contact angle of Ti-PDA and Ti-PDA@CUR1.5 was measured to test the hydrogen bond between the catechol group in CUR and PDA because the stronger the hydrophilicity, the stronger the hydrogen bond.The result shows that the order of the water contact angle is as follows: Ti (62.73 • ) > Ti-PDA (54.12 • ) > Ti-PDA@CUR1.5 (36.36 • ), which is consistent with the results presented above (Figure 4e-g).Finally, the chemical composition of CUR, Ti-PDA, and Ti-PDA@CUR1.5 was investigated by XPS, and the survey spectra prove the coexistence of Ti, C, N, and O [34,41] (Figure 4a).The high-resolution spectra of C 1s could be fit with three peaks at ca. 284.8 eV and 286.1 eV, corresponding to C-C and C-O, respectively (Figure 4b).The deconvoluted peaks at ca. 400.0 eV, 401.6 eV, and 402.8 eV can be observed in the spectrum of N 1s (Figure 4c), which are attributed to -NH=, -NH-, and -N + H-, respectively, where the -NH-bond serves as the connection between PDA and CUR [37].Additionally, C=O and C-O bonds can be detected at 531.3 eV and 533.1 eV in the spectrum of O 1s (Figure 4d).According to Yan's calculation method [33], compared with Ti-PDA, after coating CUR, the content of C-C bonds (59.4-28.1 eV) and C=O bonds (9.1-6.9 eV) decreased, and the content of -NH-bonds increased (31.8-67.2eV) in Ti-PDA@CUR1.5,further indicating that the Ti-PDA@CUR composite was successful and there was a strong interaction force between them.Finally, the water contact angle of Ti-PDA and Ti-PDA@CUR1.5 was measured to test the hydrogen bond between the catechol group in CUR and PDA because the stronger the hydrophilicity, the stronger the hydrogen bond.The result shows that the order of the water contact angle is as follows: Ti (62.73°) > Ti-PDA (54.12°) > Ti-PDA@CUR1.5 (36.36°), which is consistent with the results presented above (Figure 4e-g).E. coli and S. aureus often cause infections with surgical implants [44]; therefore, the antibacterial properties of Ti-PDA@CURx (x = 0.5, 1.0, 1.5, 2.0, 2.5) were assessed by testing against Gram-negative E. coli and Gram-positive S. aureus using a standard spread plate   (a,b) and the antibacterial rates (c,d) of Ti-PDA, Ti-PDA@CUR0.5,Ti-PDA@CUR1, Ti-PDA@CUR1.5,Ti-PDA@CUR2, and Ti-PDA@CUR2.5 against E. coli and S. aureus.

Zone of Inhibition Results
As shown in Figure 6a,b, compared with Ti-PDA, all Ti-PDA@CURx (x = 0.5, 1.0, 1.5, 2.0, 2.5) show a significant antibacterial circle.Consistent with the bacterial inhibition rate result, the diameter of the antibacterial ring shows an initial increase followed by a decrease with the increase in CUR content.More specifically, the diameter of the antibacterial circle against E. coli and S. aureus for Ti-PDA@CUR0.5,Ti-PDA@CUR1, Ti-PDA@CUR1.5,Ti-PDA@CUR2, and Ti-PDA@CUR2.5 is 9/9.3 mm, 12/10.3mm, 17/17.5 mm, 16.3/15.3mm, and 15/14.6 mm, respectively, where Ti-PDA@CUR1.5 exhibits the best antibacterial properties (Figure 6c,d).This may be because the phosphorylated groups with protonated amino groups in Ti-PDA@CUR interact electrostatically with phospholipid components of the cell membrane, potentially hindering bacteria from absorbing nutrients and multiplying [46].

Bacterial Crystal Violet Staining Results
In clinical practice, E. coli and S. aureus are frequently encountered as pathogenic bacteria, and they tend to form bacterial biofilms at the site of infection [47].In this experiment, the effects of antimicrobial susceptibility tablets on E. coli and S. aureus on bacterial biofilm formation were evaluated.As shown in Figure 7, the crystal violet color of the Ti-PDA@CUR sample became lighter compared with the Ti-PDA sample, and as the CUR content increased, it can be clearly seen that the crystal violet color of E. coli and S. aureus first lightened and then deepened.The color of E. coli in the Ti-PDA@CURx (x = 0.5, 1.0, 1.5, 2.0, 2.5) experimental groups first became lighter and then darkened.The color of S. aureus in the Ti-PDA@CURx (x = 0.5, 1.0, 1.5, 2.0, 2.5) experimental groups first became lighter and then darkened.At a loading drug concentration of 1.5 mg/mL, bacterial crystal violet was the lightest in color.This may be due to the saturation of hydrogen bond adsorption between 1.5 mg/mL CUR and Ti-PDA, which has the greatest electrostatic effect and prevents the reproduction of bacteria, so the antibacterial effect is the best.

Bacterial Crystal Violet Staining Results
In clinical practice, E. coli and S. aureus are frequently encountered as pathogenic bacteria, and they tend to form bacterial biofilms at the site of infection [47].In this experiment, the effects of antimicrobial susceptibility tablets on E. coli and S. aureus on bacterial biofilm formation were evaluated.As shown in Figure 7, the crystal violet color of the Ti-PDA@CUR sample became lighter compared with the Ti-PDA sample, and as the CUR content increased, it can be clearly seen that the crystal violet color of E. coli and S. aureus first lightened and then deepened.The color of E. coli in the Ti-PDA@CURx (x = 0.5, 1.0, 1.5, 2.0, 2.5) experimental groups first became lighter and then darkened.The color of S. aureus in the Ti-PDA@CURx (x = 0.5, 1.0, 1.5, 2.0, 2.5) experimental groups first became lighter and then darkened.At a loading drug concentration of 1.5 mg/mL, bacterial crystal violet was the lightest in color.This may be due to the saturation of hydrogen bond adsorption between 1.5 mg/mL CUR and Ti-PDA, which has the greatest electrostatic effect and prevents the reproduction of bacteria, so the antibacterial effect is the best.

Bacterial Crystal Violet Staining Results
In clinical practice, E. coli and S. aureus are frequently encountered as pathogenic bacteria, and they tend to form bacterial biofilms at the site of infection [47].In this experiment, the effects of antimicrobial susceptibility tablets on E. coli and S. aureus on bacterial biofilm formation were evaluated.As shown in Figure 7, the crystal violet color of the Ti-PDA@CUR sample became lighter compared with the Ti-PDA sample, and as the CUR content increased, it can be clearly seen that the crystal violet color of E. coli and S. aureus first lightened and then deepened.The color of E. coli in the Ti-PDA@CURx (x = 0.5, 1.0, 1.5, 2.0, 2.5) experimental groups first became lighter and then darkened.The color of S. aureus in the Ti-PDA@CURx (x = 0.5, 1.0, 1.5, 2.0, 2.5) experimental groups first became lighter and then darkened.At a loading drug concentration of 1.5 mg/mL, bacterial crystal violet was the lightest in color.This may be due to the saturation of hydrogen bond adsorption between 1.5 mg/mL CUR and Ti-PDA, which has the greatest electrostatic effect and prevents the reproduction of bacteria, so the antibacterial effect is the best.

Figure 2 .
Figure 2. UV absorption spectrum of CUR (a), the loading amount of CUR on different Ti-PDA@CUR samples (b), and the CUR standard curve (c).

Figure 2 .
Figure 2. UV absorption spectrum of CUR (a), the loading amount of CUR on different Ti-PDA@CUR samples (b), and the CUR standard curve (c).

Figure 6 .
Figure 6.Antibacterial effect (a,b) and bacteriostatic circle diameter (c,d) of E. coli and S. aureus of the samples.

Figure 7 .
Figure 7. Effect of the samples on the preformed biofilm as assessed by crystal violet staining.

Figure 6 .
Figure 6.Antibacterial effect (a,b) and bacteriostatic circle diameter (c,d) of E. coli and S. aureus of the samples.

Coatings 2024 , 12 Figure 6 .
Figure 6.Antibacterial effect (a,b) and bacteriostatic circle diameter (c,d) of E. coli and S. aureus of the samples.

Figure 7 .
Figure 7. Effect of the samples on the preformed biofilm as assessed by crystal violet staining.

Figure 7 .
Figure 7. Effect of the samples on the preformed biofilm as assessed by crystal violet staining.
• C incubator for 12 h to assess the antibacterial effectiveness of various samples against E. coli and S. aureus.