Preparation and antibacterial properties of polypropylene composite with bisphenol A-type benzoxazine.

Polypropylene (PP) is a commonly used material in medical supplies. However, its lack of antibacterial properties has led to a frequent occurrence of healthcare-associated infections that need to be addressed. This study extensively investigates the antibacterial effects of bisphenol A-type benzoxazine (BA-a), revealing a relationship between the curing degree of BA-a and its antibacterial performance. A lower degree of curing corresponds to stronger antibacterial activity. Based on this relationship, a melt blending method at 170 °C is determined to prepare PP/BA-a composite material, aiming to enhance the antibacterial properties. The antibacterial performance of PP/BA-a is evaluated by plate coating method. The results demonstrate that PP/0.8BA-a exhibits an antibacterial rate as high as 99.99% against E. coli and S. aureus. Migration experiments confirm the stability of the antibacterial effect. The extracts from PP/BA-a show no toxicity towards mouse fibroblast cells (L929). Furthermore, compared to pure PP, the addition of BA-a improves the thermal stability of PP/BA-a, with an increase of 56 °C in the onset thermal decomposition temperature (T 5% ), enabling PP to better adapt to the high-temperature environment of moist heat sterilization. This study presents a novel method for obtaining highly antibacterial medical polymer materials (PP/BA-a) with broad application prospects.


INTRODUCTION
The thermoplastic resin polypropylene FT(PP) is widely utilized in the medical field due to its exceptional chemical stability and non-cytotoxic properties [1,2].However, PP does not have antibacterial properties, which limits its application in the medical field.Biological contamination in clinical environments can result in bacteria attaching to the surface of PP and spreading to new hosts through contact [3,4], thereby endangering the lives of patients.In the United States alone, hospital-acquired infections (HAIs) are estimated to cause approximately 1.7 million infections and 99,000 related deaths each year [5].Many bacteria can survive on PP surfaces for over a month [6], posing a6 significant threat to patient health.Therefore, it is crucial to stop this transmission mode, for example, by providing materials that can directly kill bacteria upon contact.Thus, the antibacterial modification of PP holds great significance.
Standard methods for preparing antibacterial polypropylene (PP) include composite preparation, post-processing treatment, and melt blending.The composite preparation method involves the adhesion of antibacterial agents to the surface of PP, typically achieved through injection molding or compression molding.For example, adding nanosilver (Ag) to the zeolitic imidazolate framework-8 (ZIF-8) and then grafting Ag@ZIF-8 onto polypropylene melt-blown nonwoven fabric using a lamination process can achieve an antibacterial rate of 85% [7].
The post-processing treatment method involves impregnating or coating antibacterial agents onto pre-formed plastic products to provide them with surface antibacterial properties.For instance, by using the immersion-pressing-drying-plasma (IPDP) process, quaternary ammonium salt is grafted onto the surface of PP nonwoven fabric to impart good antibacterial and hydrophilic properties [8].Alternatively, fixing quaternary ammonium salt-modified chitosan (QCS) onto the polymer surface forms an antibacterial coating that enhances the antibacterial effect against Staphylococcus aureus and Pseudomonas aeruginosa by 90% [9].These methods have the advantages of using a small amount of antibacterial agents and cost-saving.However, they also have the challenges of complex processing and poor antibacterial stability.Once the surface antibacterial layer is peeled off or damaged, the antibacterial effect will be lost.
In order to obtain PP with specific antibacterial stability and easier preparation, melt blending processing method has attracted more and more attention.However, melt blending often requires a high selection of antibacterial agents, which need to have high antibacterial efficiency to ensure low dosage and good compatibility with the PP matrix.
In general, antimicrobial agents suitable for melt blending can be divided into three types: natural antimicrobial agents, inorganic antimicrobial agents, and organic antimicrobial agents.Natural antimicrobial agents are usually derived from animals and plants in nature, such as curcumin [10,11], chitosan [12], and propolis [13].This type of antimicrobial agent has advantages such as non-toxicity and strong biocompatibility, but their poor stability, low temperature resistance, and complex processing methods greatly limit their use [14,15].Inorganic antimicrobial agents usually refer to metal ions or oxides with antimicrobial activity, including Ag + [16-18], TiO2 [19], ZnO [20] and so on.Inorganic antimicrobial agents have certain advantages in terms of heat resistance, safety, antimicrobial activity, and durability, but they also have some disadvantages such as high cost and poor compatibility.Additionally, inorganic antimicrobial agents are mostly heavy metals, which may cause pollution to the ecological environment, thus causing controversy [21][22][23].
Organic antibacterial agents have the advantages of significant bactericidal effect, good compatibility, and ease of processing, but they have disadvantages such as high toxicity, poor heat resistance, and poor antibacterial stability [24].The antibacterial polypropylene composite material (PP-f) containing grafted triclosan (TCS) was prepared through melt blending process [25].The antibacterial rate of this composite material against Escherichia coli and Staphylococcus aureus exceeds 99%.This indicates that PP-f has excellent antibacterial effects.However, there is still controversy regarding the potential toxicity of TCS to the human body [26,27].Therefore, taking potential health risk into consideration, caution should be exercised when using antibacterial materials containing TCS.In order to achieve melt blending as a processing condition and overcome the disadvantages of the above-mentioned antibacterial agents, benzoxazine was chosen as the antibacterial agent.There have been many studies that have used benzoxazine as an antibacterial material and antibacterial drug.Bo et al. synthesized a cardanol-benzoxazine-based thermosetting resin containing bisphenol A dioxane (PCBOz-BD), which achieved inhibition rates of 95% and 99% against E. coli and S. aureus, respectively [28].Zafar et al. prepared Mg(II)CNSL-FA films, which showed inhibition rates of 12%, 16%, and 50% against E. coli, S. aureus, and Bacillus subtilis, respectively [29].Mohamed et al. synthesized a bio-based multifunctional benzoxazine (AP-fa-BZ), which exhibited good antibacterial activity [30].However, most of the current research is focused on the design of benzoxazine-rich molecules and the introduction of different functional groups to enhance their antibacterial properties.Currently, it has been proven that benzoxazine groups have good antibacterial performance [31,32].However, there is little research on the antibacterial performance of benzoxazine groups themselves, and there are few reports on blending them into thermoplastic resins.
In this study, Bisphenol A benzoxazine (BA-a) was chosen as the antibacterial agent, and its curing degree and antibacterial activity were investigated through infrared spectroscopy, differential scanning calorimetry (DSC), and antibacterial zone experiments.PP/BA-a antibacterial composite was prepared through melt blending, and its antibacterial performance was evaluated through in vitro antibacterial experiments.The bacterial adhesion on the surface of the material and its morphology were observed using scanning electron microscopy (SEM).The antibacterial stability of PP/BA-a was evaluated through BA-a migration experiments.The cytotoxicity of PP/BA-a towards L929 cells was evaluated through MTT assays.In addition, the crystallization and melting behavior, thermal stability and mechanical properties of PP/BA-a were studied.Table 1 summarizes the composition of all the composite materials used in this study.

Structure of BA-a and Poly BA-a
The structures of BA-a and Poly BA-a were analyzed by Fourier Transform infrared spectroscopy (FTIR) spectrometers (Thermo Fisher, USA).The scanning range is 4000 cm -1 to 400 cm -1 with a resolution of 4 cm 2 .The sample was mixed with spectral grade KBr, and the test sample was obtained by pressing.Each sample was scanned 32 times.

Isothermal curing performance of BA-a
The isothermal curing performance analysis of BA-a was carried out using a differential scanning calorimeter(METTLER, DSC3+, Switzerland).Weigh 3-5 mg sample and place it in an aluminum crucible sample dish.In the environment of nitrogen, the temperature is heated up to 300 °C from 40 °C at the rate of 10 °C /min.In order to further study the curing degree of BA-a in the actual processing process, different temperatures and times were adjusted to explore the effects of processing conditions on the curing degree.Please refer to Table 2 for specific parameters.Poly BA-a 200 180

In vitro antibacterial activity
The antibacterial activity of BA-a was explored through the inhibition zone assay.E. coli and S.
aureus of 10 6 CFU/mL were prepared, and 100 μL bacterial solution was evenly coated on Luria-Bertani (LB) solid medium.After ultraviolet sterilization, the solid sample is lightly pressed with sterilization tweezers to make it stick to the surface of the medium to ensure full contact between the sample and the medium.The liquid samples were placed on medium with the help of Oxford cups.The medium was cultured in a constant temperature incubator at 37 °C for 24 h.After the culture was completed, the medium was taken out and photographed with the camera, and the size of the antibacterial zone was measured and recorded.

In vitro antibacterial activity
PP/BA-a samples were tested for E. coli and S. aureus according to GB/T 31402-2015 test method for antibacterial properties of plastic surface.The bacterial solution with a concentration of 105 CFU/mL was prepared, and 100 μL was uniformly dropped on the surface of the sample.
Use sterilizing tweezers to gently place the sterilizing film slightly smaller than the sample surface area on the sample.Ensure that the bacterial solution is in uniform contact with the sample, and the bacterial solution does not overflow the sterilization film.The samples were placed in a constant temperature incubator and cultured at 37 °C for 24 hours.After that, the bacterial solution on the sample and sterilizing film were eluted, and then the eluent was continuously diluted by a factor of 10 with a sterile Phosphate Buffered Saline (PBS) solution.The dilute solution of 100 μL was evenly coated on LB solid medium and cultured in a constant temperature incubator at 37 °C for 18 h.Count the colonies on the petri dish, control the colonies between 30 and 300, and finally calculate the antibacterial rate based on the number of colonies.
PP and PP/BA-a samples were selected and cultured in suspensions of Escherichia coli and Staphylococcus aureus (1x10 8 CFU/mL) for 24 hours.Subsequently, the samples were fixed with a 2.5% aqueous solution of glutaraldehyde at 4 °C for more than 6 hours.The surface-unbound bacteria were then washed away with a PBS solution.Afterwards, different concentrations of ethanol (30%, 50%, 70%, 90%, 100%) were used for dehydration treatment, and finally, the bacterial morphology was observed using a scanning electron microscope (SEM).

Antibacterial stability
The antibacterial stability of PP/BA-a was evaluated according to the migration test of BA-a in PP/BA-a.Ultraviolet spectrophotometer (Shimadzu, UV-1750, Japan) was used to determine the UV absorption peaks of benzoxazine with different concentrations in ethyl acetate, ethanol and PBS, and the standard curves were drawn.PP/BA-a samples were immersed in 45ml ethyl acetate, ethanol and PBS for 1-7 days, respectively.The UV absorption peak value of the leaching solution was measured, and the migration was calculated according to the standard curve.

Cytotoxicity
The cell toxicity of PP/BA-a was tested using the MTT method.Mouse fibroblast cells (L929 cells) were seeded in a 96-well plate at a density of 4×10 3 cells per well, with each well containing 200 μL.The plate was then incubated overnight at a constant temperature of 37 °C in a CO2 incubator with 5% CO2.According to ISO 10993-12 2007 standard, the extraction of PP/BA-a was prepared, resulting in a 100% extraction sample.The sample was added to the wells of the plate, and fresh growth medium was replaced.The cells were further incubated for 1-5 days.After pretreatment, the absorbance at 570 nm was measured using an enzyme immunoassay analyzer.

Crystallization behavior
The crystallization behavior analysis of BA-a was carried out using a differential scanning calorimeter (METTLER, DSC3+, Switzerland).Weigh 3-5 mg sample and place it in an aluminum crucible sample dish.In the environment of nitrogen, the temperature is heated up to 200 °C at the rate of 50 °C /min to eliminate the heat history for 5 min.Then the temperature is reduced to 50 °C at the rate of 10 °C /min to obtain the crystallization curve.Finally, the temperature was heated to 200 °C at the rate of 10 °C/min, and the secondary melting curve was obtained.

Mechanical property
Tensile and flexural properties of the samples were measured using a universal testing machine (CMT4204, Shenzhen SANS, China).Prepare samples of 127 x 12.7 x 3.2 mm 3 and dog-boneshaped samples with 25 mm mark distance.Experiments are performed according to ASTM D790 and ASTM D638, and the final mechanical properties result is the average of at least five measurements.This indicates that BA-a cannot fully cure during the processing.In comparison to the BA-a monomer, the peak temperature of the exothermic peak decreases.This suggests that under the mentioned processing conditions, some ROP occurs in BA-a, resulting in the presence of BA-a monomers and partially ring-opened oligomers simultaneously.

Structure changes of BA-a during curing performance
In order to further explore the degree of curing of BA-a during the processing, we conducted FTIR spectroscopic analysis on it.Fig. 2(b) shows the FTIR spectra of BA-a under different processing conditions.From the curve, it can be observed that as the processing temperature increases, the characteristic peak of the oxazine group at 948 cm -1 gradually disappears, indicating an increasing degree of curing of BA-a.To analyze the ring-opening rate of the oxazine group, we calculated the degree of curing of BA-a using the change in peak area of the characteristic peaks in the FTIR spectrum.During the curing process, the -CH2 antisymmetric stretching peak at 2938 cm -1 is considered as an invariant internal reference peak, while the characteristic peak of the oxazine ring at 948 cm -1 gradually disappears with the degree of curing [35].Therefore, the degree of curing of BA-a can be calculated using equation (1).
By calculation, we can see that the BA-a ring-opening rate processed at different temperatures shows an increasing trend.The specific calculation results are shown in Table 3.In this test, Poly BA-a was tested in solid state, while BA-a was tested in solution.BA-a was dissolved in ethyl acetate as the solvent.Additionally, considering that ethyl acetate may have inherent antibacterial properties, ethyl acetate solution was chosen as the blank control.From Fig. 3, it can be observed that BA-a exhibits good antibacterial performance against Escherichia coli and Staphylococcus aureus.However, Poly BA-a has no antibacterial effect, as bacteria continue to grow around the sample without any inhibition zone.Additionally, it can be observed that a higher processing temperature results in a smaller inhibition zone.This indicates that the curing degree of BA-a can influence its antibacterial activity.Further measurements have been made to obtain the specific diameter of inhibition zone, as shown in Table 3.

Antibacterial property of BA-a
Table 3 The relationship between curing degree and antibacterial activity.
a The diameter here represents the data obtained by subtracting the diameter of the ethyl acetate control.
Table 3 illustrates the degree of curing and the diameter of the antibacterial zone of BA-a.From the Table 3, it can be observed that as the degree of curing increases, the antibacterial activity decreases.This is because the nitrogen and oxygen lone pairs on the oxazine ring enhance its reducibility, consistent with the biological effectiveness of the oxazine ring 1 .The curing process is actually the process of ROP of the oxazine ring, which results in changes in the structure of the oxazine group and the loss of antibacterial activity.Therefore, the melt blending processing temperature for PP and BA-a is set at 170 °C to minimize the occurrence of the polymerization reaction of BA-a and maintain a higher antibacterial activity.The antibacterial properties of PP/BA-a composites were evaluated by co-culture and plate coating counting.

Sample
Fig. 4 and Fig. 5 show the coating photos of E. coli and S. aureus on the plate after co-cultivation with PP/BA-a, while Fig. 6 shows the statistical graph of antibacterial rate.From Fig. 4(a), it can be seen that PP lacks antibacterial performance, as there are still 256 colonies of E. coli present in the culture dish even when the washing solution is diluted 10 2 times.Similarly, as seen from Fig. 5(a), even after diluting the S. aureus washing solution 10 2 times, there are still 118 colonies on the agar plate.However, after adding BA-a, PP gained antibacterial properties.With the increase of BA-a content, the antibacterial performance also improved gradually.From Fig. 6, it can be seen that when the addition amount is 0.4 wt%, the PP/0.4BA-acomposite material exhibits antibacterial performance, with an inhibition rate of 30.3% against E. coli and 19.7% against S.
aureus.It can be seen that compared with S. aureus, PP/BA-a composite material has a better inhibition effect on E. coli.This may be related to the structure of the two kinds of bacteria.E. coli is a typical Gram negative bacterium, while S. aureus is a typical Gram positive bacterium.The dense skin polysaccharide layer on the outer layer of Gram positive bacteria can serve as a barrier protection, preventing the addition of antibacterial agents into cells and causing damage to the cytoplasm [36,37].Gram negative bacteria lack such a dense protective layer, which may be the reason why BA-a has better antibacterial effects on E. coli.When the addition amount reached 0.8 wt%, the antibacterial effect of PP/0.8BA-a composite against E. coli and S. aureus could reach 99.9%, showing excellent antibacterial performance.Moreover, through SEM images, it can be observed that a large number of E. coli adhere to the surface of untreated PP, as shown in Fig. 7(a), and they appear rod-shaped.Similarly, Fig. 8(a) reveals that a large number of S. aureus adhere to the surface of PP, and they appear spherical.
However, after adding 0.4 wt% BA-a, it can be seen from Fig. 7(b) and Fig. 8(b) that the E. coli and S. aureus on the surface of PP/0.4BA-a start to rupture and deform, but some of their shapes still remain intact, which is consistent with the antibacterial results.Moreover, Fig. 7(c) and Fig. 8(c) show no adhesion of E. coli or S. aureus on the surface of PP/0.8BA-a, indicating that PP/0.8BA-a not only possesses good antibacterial performance but also inhibits bacterial adhesion.
In addition to antibacterial properties, antibacterial stability is also very important for materials.
It has been proved that PP/BA-a has excellent antibacterial properties, and the antibacterial stability will be further explored in the next step.The PP/ 0.8BA-a samples were soaked in ethyl acetate and ethanol for 7 days, and the leaching of BA-a on each day was detected by UV to characterize the migration of the antibacterial agent.
Fig. 9 Migration of BA-a in different solvent.
Fig. 9 was chosen as the solvent because BA-a has a better solubility in ethyl acetate.It can be seen that the migration of BA-a in ethyl acetate reaches the highest on the seventh day and is still less than 1%, indicating a low migration.This is because of the good compatibility of BA-a and PP.The choice of ethanol as solvent is to simulate the disinfection scene in daily life; The choice of PBS as the solvent is to simulate the scenario of daily use.It can be seen that BA-a has little migration in ethanol and PBS.Among them, the migration in ethanol solvent is slightly higher than that in PBS solvent, which is because ethanol solvent is an organic solvent.The low migration also indicates that PP/0.8BA-a has strong antibacterial stability.In general, PP/0.8BA-a composite has good antibacterial properties and antibacterial stability, which indicates that the composite has a good application prospect.
In addition to antibacterial performance and antibacterial stability, the potential toxicity of organic antibacterial agents to the human body has always been a topic of great concern.Therefore, we conducted a cytotoxicity test.According to the results of the MTT experiment, the relative activity of L929 cells co-cultured with 100% extract of PP/0.8BA-a for 1-5 days was above 98% (Fig. 10).According to the GB/T 16886.5-2017standard, no significant decrease in cell proliferation was observed in the extract, indicating that the extract has a cell toxicity level of 1 and will not cause harm to the human body.
This result indicates that the PP/BA-a extract is non-toxic and can be used in medical materials.

Physical properties of PP/BA-a
Fig. 11 The DSC curves of PP/BA-a: (a) DSC cooling curves; (b) DSC heating curves.
Table 4 The relationship between curing degree and antibacterial activity.
Fig. 11 and Table 4 is the crystallization data of PP/BA-a, in which the crystallinity is tested and calculated using the following equation ( 2) ΔHf------PP enthalpy of complete melting, which is 209 J/g [38].
It can be seen that the addition of BA-a has no great influence on the crystallization behavior of PP.The melting temperature and crystallization temperature did not change much.However, the enthalpy of secondary melting and the crystallinity are increased.Moreover, the addition amount has little influence on the crystallinity, no matter the addition of 0.2 wt% or 1 wt%, the crystallinity is increased from 33% to around 36%, which is 3% higher than neat PP.Table 5 Thermal properties of PP/BA-a.
The PP/BA-a composite was analyzed by thermogravimetric analysis.The addition of BA-a has a certain effect on the thermal stability of PP.Fig. 12 shows the TG curve and the DTG curve.The temperatures at 5% weight loss (T5%), 10% weight loss (T10%), 10% weight loss (T10%) and maximum weight loss (Tmax) are shown in Table 5.
As a thermosetting resin, BA-a has good thermal stability.From Fig. 12(c), it can be seen that the TG curve of BA-a shows the first weight loss occurring at approximately 240 °C, consistent with the DSC results.This is because the monomer undergoes ROP process and releases CO2 and some low molar mass compounds.When comparing BA-a and Poly BA-a with PP, it is found that both BA-a and Poly BA-a have lower weight than PP before 400 °C, but higher weight than PP after 400 °C, and there is still about 40% residue at 600 °C.However, as shown in Fig. 12(a), with the addition of BA-a, PP/BA-a shows improved thermal stability compared to pure PP.This is because BA-a partially opens the ring, converting the oxygen atoms on the imidazole ring into phenolic hydroxyl groups.The phenolic hydroxyl groups combine with the carbon chain free radicals in PP, inhibiting the auto-oxidation process of the polymer and improving its thermal stability [39,40].With the increase in the amount of BA-a addition, the initial decomposition and the initial decomposition temperature(T5%) increases by 56 °C compared to PP, while the initial decomposition temperature of PP/1.0BA-a decreases slightly.This may be because when the BA-a addition amount is less than 0.8 wt%, the phenolic hydroxyl group dominates the improvement of PP thermal stability, while when the BA-a addition amount exceeds 0.8 wt%, the poor thermal stability of BA-a itself leads to a decrease in the initial decomposition temperature of PP/BA-a.The addition of 0.8 wt% BA-a is also the saturation point for antibacterial performance of the material.Therefore, PP/0.8BA-a exhibits excellent antibacterial performance and better thermal stability.It is suitable for medical materials and can better withstand hightemperature sterilization scenarios such as dry heat and moist heat.decrease is very small.The flexural modulus has a small amplitude fluctuation, and there is no obvious upward or downward trend.It can be seen that BA-a has no great influence on the mechanical properties of PP.In summary, both PP/BA-a and PP possess excellent mechanical properties, making them suitable for various applications in medical materials.

CONCLUSIONS
This study investigated the antibacterial modification of a common medical material, PP.By blending bisphenol A benzoxazine (BA-a) with PP through melt blending, a new material, PP/BAa, was obtained.It has been confirmed that BA-a exhibits good antibacterial activity, which is influenced by the degree of curing.Higher curing degree of BA-a leads to lower antibacterial activity.Therefore, a processing temperature of 170 °C was determined, and PP/0.8BA-a showed an antibacterial activity of 99.99% against both gram-negative and gram-positive bacteria.
Additionally, migration tests revealed that PP/BA-a possesses good antibacterial stability.MTT experiments confirmed the non-toxicity of PP/BA-a extracts to L929 cells.Furthermore, TGA experiments verified the improved thermal stability of PP/BA-a, with an initial decomposition temperature increase of 56 °C compared to PP.This study presents a valuable melt blending method for the preparation of medical-grade antibacterial modified PP materials, enhancing their thermal stability.The method exhibits promising potential for practical applications.

Fig. 1
Fig.1FTIR spectra before and after BA-a polymerization.

Fig. 2
Fig. 2 (a) DSC curves of BA-a under different processing conditions and Poly BA-a; (b) FTIR

Fig. 10
Fig. 10 Results of the cytotoxicity of BECs in vitro of PP/0.8BA-a.

Fig. 12 (
Fig. 12 (a)TG curve of PP/BA-a (b)DTG curve of PP/BA-a.(c)TG curve of BA-a and Poly BA-a;

Fig. 13
Fig. 13 shows the t mechanical properties of PP/BA-a, including tensile strength and flexural

Table 1
Compositions of PP/BA-a composite materials.