A-D-A covalent organic-inorganic heterojunctions with enhanced photocatalytic performance for robust anti-bacteria

Background: Organic semiconductors have attracted much attention due to their excellent biocompatibility, tunable electronic structure, low cost, and antimicrobial phototherapy. However, owing to the high exciton binding energies, organic semiconductor is constrained by their short exciton diffusion length, leading to inefficient transportation of photogenerated carriers and deficient antibacterial capability. Methods: To address this issue, a quad-channel synergistic antibacterial nano-platform of copper sulfide/organic semiconductor (CuS/IEICO-4F) heterojunctions with enhanced photocatalytic performance is designed and manufactured, which can produce localized heat and raise the levels of extracellular reactive oxygen species under near-infrared laser irradiation. Simultaneously, the released Cu 2+ can consume intrabacterial glutathione, destroying the defense system and ultimately leading to bacterial inactivation. Results: In vitro antibacterial experiments demonstrate that the organic-inorganic bio-heterojunctions possess the potent antibacterial capacity and effective bacterial eradication. Conclusion: This countermeasure shows great promise for application in infectious wound regeneration.


Background
According to the World Health Organization (WTO), tens of millions of patients die every year from bacteria-related infections, posing a serious threat to human lives and property [1,2].In the current healthcare system, antibiotics are a common and efficient remedy, whereas the overuse of antibiotics has led to a growing problem of bacterial resistance [3].Therefore, searching for a non-antibiotic therapy has become one of the current research hotspots [4].
Phototherapy as an antibiotic-free treatment with the advantages of spatiotemporal selectivity, non-invasiveness, low side effects, and broad-spectrum antimicrobial resistance, is considered a promising candidate for anti-pathogens [5].Photosensitizers, as the key to phototherapy, can generate ROS through light excitation to kill microorganisms by reacting with oxygen (O 2 ) and water (H 2 O) [6].Compared to traditional inorganic photosensitive antimicrobial materials such as Graphene Oxide and Au NPs, organic photosensitizers such as hydrogels and indocyanine green are widely used in the field of bio-antimicrobial remediation due to their superior biocompatibility, adhesion, and biodegradability [7][8][9][10].Organic semiconductors as a new type of organic photosensitizer have been widely studied in antimicrobial phototherapy owing to the special Acceptor-Donor-Acceptor (A-D-A) conjugated structures, which ensure strong light absorption and photothermal conversion in the NIR region [11,12].The lower bandgap contributes to the generation of single-linear oxygen ( 1 O 2 ), thus contributing to the improvement of photodynamic therapy (PDT) efficacy [13,14].However, organic semiconductors often have high exciton binding energies and long diffusion lengths, resulting in poor carrier transport and inefficient charge production, which inhibits their photocatalytic properties [15].Therefore, increasing the efficiency of charge migration and enhancing the photocatalytic activity is an immersive desire to establish a directional channel that can extend the exciton diffusion length, allowing them to move more easily from the semiconductor to the heterogeneous interface.
Over the past decades of years, heterogeneous interface engineering has endowed materials with photo-/sono-/microwave-induced capability, making them useful for disease treatment such as pathogenic infections and cancer [16,17].The potential mechanism is that heterogeneous interface engineering inhibits the complexation of electron-hole pairs, which enhances the efficiency of carrier utilization and facilitates the enhancement of photodynamic effects [18].Synergistic photothermal therapy (PTT) can effectively inhibit the growth and spread of bacteria or cancer cells [19].It has been shown that all-organic semiconductor heterojunctions C3N4/PDINH photocatalysts have greatly improved light absorption as well as the mobility and separation of photogenerated carriers, and exhibit excellent bactericidal effects and biocompatibility [20].Despite their potential for biological applications, all-organic semiconductor heterojunctions currently face issues with stability, conductivity, and long-term stability [21].CuS, being classified as a p-type semiconductor, possesses a narrow band gap and demonstrates noteworthy photoinduced thermal therapy (PTT) or photodynamic therapy (PDT) effects when exposed to light with varying wavelengths [22].Furthermore, as one of the vital slight elements, Cu 2+ performs numerous vital physiological and pharmacological tasks for the body.Some researchers have indicated that Cu 2+ can interact with different proteins and enzymes within the body to take part in metabolic activities [23].Moreover, it can encourage the formation of melanin, which is advantageous for wound healing [24,25].
Based on this, we propose a strategy for fabricating A-D-A conjugated organic-inorganic bio-heterojunctions with improved photocatalytic performance via in situ growth of IEICO-4F to form a tight heterogeneous contact interface on the surface of hollow CuS NPs, which confers robust antimicrobial remediation capability.By regulating the interfacial energy band structure through close contact with inorganic semiconductor materials and constructing a directional channel for electron transfer, the transport efficiency of photogenerated carriers in organic semiconductors is improved for robust antibacterial in infectious wound healing.As shown in Figure 1A, CuS/IEICO-4F bio-heterojunctions, and CuS hollow microsphere NPs were synthesized via in situ recrystallization and hydrothermal Under NIR-II irradiation, the CuS/IEICO-4F bio-heterojunctions not only possess favorable PTT properties but also efficiently exploit the infected microenvironment through enhanced PDT and chemodynamical therapy (CDT) effects, resulting in enhanced ROS yield.Furthermore, the Cu 2+ released from CuS/IEICO-4F bio-heterojunction in the infected microenvironment can efficiently consume GSH and destroy the bacterial redox (Figure 1B).In vitro antimicrobial experiments demonstrate that CuS/IEICO-4F bio-heterojunctions can eradicate bacteria by virtue of PTT, peroxidase (POD)-like property, and GSH peroxidase (GPx)-like property.Overall, this study provides a novel strategy for enhancing photocatalytic therapy against bacterial infections.

Synthesis of hollow CuS NPs
30 mg of L-cysteine was dissolved in 50 mL of copper sulfate solution, with a concentration of 0.01 M. To this solution, 50 mL of CH 3 CSNH 2 solution, at a concentration of 0.02 M, was added slowly while continuously stirring for 30 minutes.The resulting mixture was then transferred to a Teflon-lined stainless-steel autoclave, placed in an explosion-proof box, and heated at 200 °C for 20 hours.After completion of the reaction, the mixture was allowed to cool down to room temperature naturally and then washed three times each with ethanol and water.The resulting black powder was dried at 60 °C for an hour, and it was identified as CuS NPs.

Synthesis of CuS/IEICO-4F Bio-Heterojunctions
CuS/IEICO-4F were created by the straightforward in situ recrystallization process.10 mg of IEICO-4F was added to 50 mL of chloroform (CHCl 3 ) and vigorously agitated for 20 min until a clear solution was obtained.Subsequently, 1 g of CuS was added to the IEICO-4F solution, sonicated for 30 min, and stirred for 12 h at room temperature before being raised to 65 °C to completely evaporate the chloroform.Ultimately, the produced black powder was dried for one to two hours at 70 °C in an oven.In the end, CuS/IEICO-4F containing 0.2 weight percent of IEICO-4F was created.Characterization SEM (JSM7610F, JEOL) is employed for analyzing the surface morphology and constituent elements of as-prepared materials.XRD (EMPYREAN, PANalytical) is employed for characterizing the crystal structure of materials.

Measurement of extracellular ROS
The existence of 1 O 2 produced by materials under NIR irradiation was detected using 1,3-diphenylisobenzofuran (DPBF) as the 1 O 2 -trapping agent.Normally, the DPBF solution was mixed with different sample solutions and irradiated with a 10 min NIR laser.Retain supernatant after irradiation, a UV-vis spectrophotometer (UV-1800PC, AOELAB, China) was applied to detect the absorbance.
The Methylene blue (MB) degradation experiment was used to verify the production of hydroxyl radicals (•OH) under NIR-Ⅱ irradiation.In a nutshell, different as-prepared sample solutions and MB solutions were mixed and co-incubated under dark conditions for 30 min.After incubation, the irradiation group was exposed to a near-infrared laser for 10 min and retained the supernatant.and the UV-vis absorbance value of each group of sample solutions was measured.

GPx-like property detection
The materials were combined with a solution of GSH (400 µL, 0.8 mM) and allowed to incubate in a dark environment for 15 minutes.Following this, the mixtures underwent irradiation using a NIR laser (1.5 W•cm −2 , 10 min) or were subjected to treatment in the absence of light.After the process, the Tris buffer (450 µL, pH = 8) and 5,5'-Dithiobis (2-nitrobenzoic acid) (DTNB) solution (120 µL, 10 mM) were added into the as-prepared mixed solution.The peak absorption was detected at 410 nm.H 2 O 2 and PBS were used as positive control and negative control, respectively.The GSH consumption rate (β%) was calculated according to the following equation: β% =(ODNEG-ODPOS-ODSPL)/(ODNEG-ODPOS)×100% (1) ODSPL, ODNEG, and ODPOS are the peak absorption values of different materials, negative controls, and positive controls, separately.

In vitro antibacterial activity
For this experiment, Staphylococcus aureus (S. aureus) and Escherichia coli (E.coli) were used as representative models of Gram-positive and Gram-negative germ strains, respectively.Bacteria suspensions, each containing 1.0 × 10 4 CFUs and measuring 800 µL, were mixed with sample solutions prepared beforehand (200 µL, 2000 µg/mL) in a 48-well plate.The mixed solutions were then subjected to NIR laser irradiation (1.5 W•cm −2 ) for 10 min.The dark control groups were also included without the irradiation.Afterward, each sample was plated onto solid Luria-Bertani (LB) agar plates and cultured overnight at 37 °C.The number of colonies was then counted.For statistical accuracy, all the experiments were repeated three times.The antibacterial rate (γ%) was calculated using the following equation: γ% = (CFUNEG -CFUSPL)/CFUNEG × 100% (2) CFUSPL and CFUNEG are the colony-forming units of different samples and negative controls, respectively.

Statistical analysis
The data is presented as the mean ± standard deviation (SD).To compare multiple groups, either one-way ANOVA or two-way ANOVA was used, followed by Tukey's multiple comparison test, with GraphPad Prism 8.0 software (GraphPad Software, Inc., La Jolla, USA).Values of * P < 0.05 and ** P < 0.01 were denoted statistically significant, respectively.

Characterization of organic-inorganic Bio-Heterojunctions
The X-ray diffraction (XRD) pattern of the prepared materials is obtained and analyzed.As seen in Figure 2A, the characteristic peaks observed at 2θ values of 29.27°, 31.78°, and 47.94°are assigned to the (102), (103), and (110) crystalline planes of CuS (PDF#06-0646), respectively.It is observed that the addition of the organic semiconductor IEICO-4F had little effect on the crystallinity of CuS.The surface morphology of the prepared materials was observed using SEM.As shown in Figure 2B, 2C, CuS has a hollow microsphere structure, while IEICO-4F is an amorphous crystal.Figure 2D shows the surface of CuS hollow microspheres, which have an amorphous organic semiconductor crystal, IEICO-4F, attached to them.The EDS spectrum of CuS/IEICO-4F bio-heterojunctions (Figure 2E) indicates that the sample possesses the expected elemental combination.Additionally, the elemental mapping image (Figure 2F) reveals that the materials' elements including Cu, C, N, O, F, and S are uniformly Submit a manuscript: https://www.tmrjournals.com/bmecdistributed throughout the material.Based on the results obtained, it can be inferred that CuS/IEICO-4F bio-heterojunction has been successfully synthesized at the initial stage.

Photothermal performance
CuS and IEICO-4F both have been considered to process undiminished photothermal ability under NIR laser, thus the photothermal properties of the studied materials were measured.As shown in the temperature change curves of various materials (Figure 3A), After being irradiated by NIR for 10 min, the different samples (CuS, IEICO-4F, and CuS/IEICO-4F) possess a significant increase in temperature.The highest temperature rise is observed in CuS, which increased by about 45 °C.However, such high temperatures could potentially damage the surrounding tissues around the wound.In contrast, IEICO-4F shows poor dispersion in water due to its hydrophobic nature.The CuS/IEICO-4F group can reach the sterilization temperature without causing any damage to the wrinkled tissue of the wound.Enhancement of the photothermal performance of CuS/IEICO-4F bio-heterojunctions can be achieved by increasing NIR power and sample concentration, as shown in Figure 3B, 3C.Moreover, we also recorded the temperature change curves of CuS/IEICO-4F under both light and non-light cycles to investigate its photothermal stability.The results show that CuS/IEICO-4F exhibits no significant temperature change during 4 cycles.The maximum temperature is maintained at around 55 °C, while the minimum temperature fluctuates slightly around 27 °C, indicating excellent photothermal stability (Figure 3D).

Photodynamic and chemodynamical properties
Apart from the photothermal property, the photodynamic effect of samples was evaluated using MB to explore the production of •OH.As depicted in Figure 4A, both the CuS and CuS/IEICO-4F groups exhibit a significant reduction in absorbance without NIR irradiation.However, there was no noticeable difference between the two material groups.On the other hand, the absorbance of the IEICO-4F group did not change significantly from that of the control group.It can be concluded that the decrease in MB absorbance is primarily attributed to the chemodynamical effect of CuS.When exposed to NIR light, the IEICO-4F group appears dark, as seen in Figure 4B.The CuS group shows a significant decrease in absorbance, suggesting that it produces a large amount of •OH due to the combined photodynamic and chemodynamical forces.In contrast, the absorbance of the CuS/IEICO-4F group shows a rewarding decrease.It indicates that the formation of heterojunction has greatly enhanced the photocatalytic effect of the material, leading to a synergistic effect in addition to the chemodynamical effect possessed by CuS.As a result, the yield of •OH has been dramatically increased.
After proving the existence of •OH, DPBF was selected as a 1 O 2 trapping agent to demonstrate the presence of the produced 1 O 2 which will decrease the absorption intensity centered around 410 nm.As depicted in Figure 4C, the IEICO-4F group exhibited only a slight decrease in DPBF absorbance after NIR laser exposure, indicating low DPBF degradation.As a result of the photocatalytic effect of the CuS, there was only a moderate decrease in the absorbance of DPBF.As opposed to that the heterojunction group experienced the greatest decrease in absorbance, indicating that CuS/IEICO-4F was able to produce more 1 O 2 under NIR laser.Figure 4D demonstrates that the photodynamic performance of the CuS/IEICO-4F heterojunction was significantly strengthened under NIR laser irradiation with the prolongation of exposure time.The construction of the heterojunction material system resulted in this improvement.
Previous literature has reported that Cu and Fe-containing samples exhibit POD-like activities, catalyzing substrate oxidation via Fenton-like reactions.In this part of the experiment, the POD-like activity was explored using TMB as the substrate in the presence of H 2 O 2 without NIR laser irradiation.As shown in Figure 4E, the photographs of the IEICO-4F group did not exhibit a distinct blue color consistent with its absorbance curve, indicating the absence of POD-like activity for IEICO-4F.However, compared to the IEICO-4F group, the TMB solutions in both the CuS and CuS/IEICO-4F groups showed significant colorations, with the absorbance curves showing a distinct characteristic peak of absorption around 650 nm.The results demonstrate that CuS acts as a catalyst for the formation of •OH from H 2 O 2 via a Fenton-like reaction, which subsequently oxidizes TMB to form blue ox-TMB.The absorbance curves in Figure 4E indicate that the CuS/IEICO-4F group exhibits a greater characteristic peak intensity compared to the CuS group.This implies that the CuS NPs in the hollow microsphere display an improved POD-like activity owing to the construction of CuS/IEICO-4F heterojunctions.CuS/IEICO-4F has the potential to significantly enhance antibacterial properties through its enhanced POD-like activity in acidic infectious microenvironments.The experimental results strongly suggest that this technology can be a game-changer in the field of antibacterial treatments.In a word, the CuS/IEICO-4F bio-heterojunctions have superior photocatalytic properties and ROS yields, such as •OH and 1 O 2 , within the infected microenvironment.These ROS work in synergy with the POD-like activity, making it a promising treatment option for robust bactericidal applications.

GSH depletion
GSH is a crucial component in pathogenic bacteria's resistance to oxidative stress damage and helps to maintain normal intracellular redox homeostasis.However excessive ROS and certain metal ions (such as Fe (III) and Cu (II)) deplete GSH in the bacterium, which  causes the antioxidant defense system to malfunction and speeds up the pathogen's demise.Hence, consuming adequate amounts of GSH is crucial for the successful elimination of bacteria.To determine the amount of GSH used by each group of materials, DTNB was selected as the GSH color developer.Attributable to the reaction between DTNB and GSH produces a yellow 5-thio-2-nitrobenzoic acid, which has its peak absorption value at 412 nm.This property makes it convenient to measure the consumption of GSH by each group.The reaction of DTNB with the sulfhydryl group of GSH can produce yellow 5-thio-2-nitrobenzoic acid, which has the maximum absorption peak at 412 nm.Therefore, to study the consumption of GSH by each group of materials, DTNB was chosen as the GSH color developer.As shown in Figure 5A, there was no significant change in solution color in the IEICO-4F group with or without light, indicating that the material was not ideal for GSH consumption.Since CuS catalyzes the oxidation of organ thiols (R-SH) to disulfides (RSSR), the color of the solution was lightened in the CuS group.Based on the color comparison and quantification of CuS between the light and non-light groups (Figure 5B), it can be inferred that the photocatalytic effect of CuS is compromised due to the complexation of electron-hole pairs.As a result, the production of ROS is reduced, which in turn fails to achieve the desired consumption of GSH.Interestingly, the CuS/IEICO-4F group faded significantly to almost colorless under light conditions.The group exhibited a GSH consumption rate of around 60%, which was higher than the corresponding GSH consumption rate of the dark group, which was only about 40%.This indicates that under NIR irradiation, the CuS/IEICO-4F bio-heterojunctions produced more ROS, which synergized with Cu (II) to further increase GSH consumption.The CuS/IEICO-4F bio-heterojunctions have a strong ability to consume GSH, demonstrating its effectiveness.The experimental results indicated that CuS/IEICO-4F can enhance GSH consumption, disrupt bacterial redox balance, cause oxidative stress, and improve bacterial killing ability.

In vitro antibacterial activities
The above experiments confirm that the CuS/IEICO-4F heterojunction has excellent photothermal, photodynamic, and chemodynamical properties and favorable GSH consumption.To investigate the sterilization effect of various samples against S. aureus and E. coli.The flatbed coating method was conducted, the number of bacterial colonies was counted (Figure 6A, 6C) and the antibacterial rate was calculated (Figure 6B, 6D).A large number of bacterial colonies can be observed in all dark control groups.Differently, CuS groups show their good antibacterial ability against both S. aureus and E. coli with NIR irradiation, due to the extraordinary photothermal property and antimicrobial effect of Cu 2+ ions of CuS.On the contrary, the antibacterial rate of IEICO-4F groups drops to 49% and 45% against S. aureus and E. coli, separately.The antibacterial results are consistent with the photothermal performance results.Moreover, the antibacterial rate subsequently reached 100% against S. aureus and 99% against E. coli in organic-inorganic CuS/IEICO-4F bio-heterojunctions groups, respectively.These results indicate that organic-inorganic CuS/IEICO-4F bio-heterojunctions exhibit outstanding antimicrobial properties in vitro.

Conclusion
In summary, we constructed an A-D-A conjugated organic-inorganic bio-heterojunction nanoplatforms with an enhanced photocatalytic performance by the in situ recrystallisation method.Under NIR irradiation, the CuS/IEICO-4F heterojunction possesses favorable photothermal properties for local heat and photocatalytic property for ROS yield as well as released Cu 2+ for GPx-like property, resulting in an effective elimination against bacteria by disrupting their redox homeostasis.In vitro antimicrobial experiments demonstrate the robust and effective antimicrobial capacity of the CuS/IEICO-4F bio-heterojunction, which presents great potential for application in infectious skin wounds.The countermeasure provides a novel approach for enhanced photocatalytic therapy against infections.

Figure 3
Figure 3 Photothermal experiments.(A) Temperature change curves of different materials; (B) Temperature change curves of CuS/IEICO-4F under irradiation of NIR light with different power; (C) Temperature change curves of CuS/IEICO-4F with different concentrations; (D) Photothermal cycling curves of CuS/IEICO-4F.

Figure 4
Figure 4 Photodynamic performance tests.(A) MB absorbance changes caused by •OH produced by IEICO-4F, CuS, and CuS/IEICO-4F in the absence of NIR irradiation; (B) MB absorbance changes caused by •OH produced by IEICO-4F, CuS, and CuS/IEICO-4F in the presence of NIR irradiation; (C) IEICO-4F, CuS, CuS/IEICO-4F absorbance changes induced by the generation of 1 O 2 under NIR irradiation; (D) degradation curves of DPBF induced by the generation of 1 O 2 under NIR irradiation of CuS/IEICO-4F over time; (E) UV-vis spectra of TMB solutions in the presence of H 2 O 2 without NIR laser irradiating samples of each material (TMB color change for the corresponding group is shown in the inset).causesthe antioxidant defense system to malfunction and speeds up the pathogen's demise.Hence, consuming adequate amounts of GSH is

Figure 5
Figure 5 GSH depletion experiments.(A) Photographs of the color change of GSH solutions after reaction with various materials with or without NIR light irradiation; (B) Corresponding GSH depletion rate.

Figure 6
Figure 6 In vitro antimicrobial performance experiments.Results of smear plate method of different samples against S. aureus (A) and E. coli (C); Counts of colonies corresponding to S. aureus (B) and E. coli (D).