A microneedle patch that uses cyanin-derived pH-responsive photofunctional molecules to inhibit bacterial infection in wounds

Abstract

This invention proposes a microneedle patch that seamlessly integrates infection-activated bioluminescence imaging, photodynamic therapy, and synergistic antibacterial activity with carbon monoxide gas to inhibit bacterial infection in wounds using anthocyanin-derived pH-responsive photofunctional molecules. The invention presents a high-performance phototherapy agent based on anthocyanin derivatives that can sense and dynamically adjust its fluorescence and photodynamic therapy (PDT) properties in response to changes in acidity within the infection microenvironment. Subsequently, a hydrophobic probe is co-assembled with a reactive oxygen species (ROS) CO donor to form a biocompatible therapeutic nanosystem, which is further encapsulated in a hyaluronic acid microneedle patch to enhance its mechanical penetration and controlled-release activity at the infection site. The binding of PDT with the CO gas cascade produces a powerful bacterial elimination effect, significantly inhibiting bacterial biofilm-mediated infection. This ingenious combination of infection-activated bioimaging and synergistic antibacterial therapy offers significant potential for the effective and personalized management of infection-related diseases.

Description

Technical Field

[0001] This invention belongs to the field of medical materials technology. Background Technology

[0002] Currently, antibiotic treatment forms the cornerstone of bacterial infection management. However, prolonged exposure to antibiotics can ultimately lead to increased bacterial resistance, and in some cases, even multidrug-resistant (MDR) strains. Furthermore, overuse of antibiotics can cause a range of adverse effects, including acute and chronic damage to the liver and kidneys. Therefore, there is an urgent need for alternative antimicrobial strategies that can address bacterial infections with high precision and efficiency.

[0003] In current antibacterial strategies, photodynamic therapy (PDT), as a non-invasive treatment technique, has become a powerful approach to addressing the challenges of bacterial infections. PDT utilizes the reaction of photosensitizers with oxygen under light irradiation to generate cytotoxic reactive oxygen species (ROS), which can cause oxidative damage to bacterial cell walls, lipids, proteins, and nucleic acids, ultimately leading to bacterial death. Compared to traditional antibiotic treatment, PDT has the advantages of broad-spectrum efficacy and high spatiotemporal control, and can be repeated as needed without inducing treatment resistance. Due to these unique advantages, PDT has been used clinically to treat refractory localized infectious diseases, showing the potential to delay the development of multidrug resistant (MDR) diseases caused by frequent switching of antibiotics.

[0004] While photodynamic therapy (PDT) offers a valuable alternative for treating infections, it's worth noting that several challenges remain that significantly limit its efficacy. PDT has demonstrated high antibacterial efficiency and a broad spectrum of bacteria in vitro. However, in most cases, current trials require the in-situ application of high doses of photosensitizers, such as spraying onto wounds, to completely kill bacteria. Moreover, if mishandled, excessive photosensitizers can inevitably damage healthy host tissue. Furthermore, most reported photosensitizers are in a "constantly lit" state, regardless of whether they are located within the infection lesion, lacking adaptability to different infection severities. Typically, infection sites contain complex tissue microenvironments, and non-specific accumulation of photosensitizers in normal cells or surrounding tissues can lead to serious side effects. Another issue associated with PDT for infection treatment is the inherent oxygen dependence of PDT and the limited permeability of irradiated light in tissues, which greatly reduces its effectiveness in eradicating bacteria. Therefore, integrating phototherapy with other treatment modalities would be a good option to improve antibacterial efficacy. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a microneedle patch that utilizes cyanin-derived pH-responsive photofunctional molecules to inhibit bacterial infection in wounds.

[0006] The technical solution adopted in this invention is as follows:

[0007] The structural formula of the cyanin-derived pH-responsive photofunctional molecule (CyOH) in this invention is as follows:

[0008]

[0009] The preparation method of CyOH is as follows: 1.5 mL of 2,3,3-trimethylindole is dissolved in 17 mL of acetonitrile. After repeated deoxygenation three times, 1.0 g of 3-iodo-1-propanol is added to the above system and heated under nitrogen protection under reflux for 24 h. After the reaction system is cooled to room temperature, the solvent is removed by vacuum distillation. The product is then washed with petroleum ether, and the petroleum ether is removed by rotary evaporation. It is then dissolved in a small amount of acetonitrile, poured into cooled diethyl ether, and the precipitate is precipitated and filtered to obtain 1-(3-hydroxypropyl)-2,3,3-trimethyl-3H-indole.

[0010] Subsequently, 1-(3-hydroxypropyl)-2,3,3-trimethyl-3H-indole and 4-[bis(4-methoxyphenyl)amino]benzaldehyde were dissolved in 15 mL of acetonitrile solution. Piperidine was added dropwise, and the reaction system was heated to 80 °C and reacted under nitrogen protection for 10 h. The product was obtained after purification.

[0011] A microneedle patch that uses cyanin-derived pH-responsive photofunctional molecules to inhibit bacterial infection in wounds, wherein each microneedle in the microneedle patch has a 500μm×500μm square base, each microneedle gradually tapers from the base to the tip, with a height of 1000μm, the microneedles are arranged in a 10×10 array, and the tip-to-tip spacing is 900μm.

[0012] The tip is molded from HMHA hydrogel containing 10 mg / mL CyOH & CO Nps, and the base is molded from LMHA hydrogel containing 10 mg / mL CyOH & CO Nps; the HMHA hydrogel has a concentration of 500 kDa, and the LMHA hydrogel has a concentration of 10 kDa.

[0013] The preparation method of CyOH&CO Nps is as follows: Add 8.0 mg CyOH, 2.0 mg CORM-401, 40 mg DSPE-PEG, and 10 mL of water to 1.0 mL DMSO. Stir rapidly at 1400 r / min, insert a needle below the liquid surface, and slowly add the solution dropwise over five minutes. Dialyze for 4 hours, changing the water every half hour to obtain CyOH&CO NPs.

[0014] This invention also provides a method for preparing a microneedle patch that utilizes cyanin-derived pH-responsive photofunctional molecules to inhibit bacterial infection in wounds, the specific steps of which are as follows:

[0015] 1) Dissolve 1.5 mL of 2,3,3-trimethylindole in 17 mL of acetonitrile, remove oxygen three times, add 1.0 g of 3-iodo-1-propanol to the above system, and heat under reflux for 24 h under nitrogen protection; after the reaction system is cooled to room temperature, remove the solvent by vacuum distillation; then wash the product with petroleum ether, remove the petroleum ether by rotary evaporation, dissolve it in a small amount of acetonitrile, pour it into cooled diethyl ether, precipitate the precipitate and filter to obtain 1-(3-hydroxypropyl)-2,3,3-trimethyl-3H-indole;

[0016] Subsequently, 1-(3-hydroxypropyl)-2,3,3-trimethyl-3H-indole and 4-[bis(4-methoxyphenyl)amino]benzaldehyde were dissolved in 15 mL of acetonitrile solution. Piperidine was added dropwise, and the reaction system was heated to 80 °C and reacted under nitrogen protection for 10 h. The purified product was denoted as CyOH.

[0017] 2) Add 8.0 mg CyOH, 2.0 mg CORM-401, 40 mg DSPE-PEG, and 10 mL of water to 1.0 mL DMSO. Stir rapidly at 1400 r / min, insert the needle below the liquid surface, and slowly add the solution dropwise over five minutes. Dialyze for 4 hours, changing the water every half hour to obtain CyOH & CO NPs.

[0018] 3) A polydimethylsiloxane PDMS micromold with rectangular conical holes is created by pouring a premixed PDMS solution into a master mold, followed by degassing (in a vacuum oven for 15 minutes) and curing (at 65°C for 5 hours). The mold is designed according to the following rules: each microneedle has a 500μm × 500μm square base, each microneedle gradually tapers from the base to the tip, with a height of 1000μm, and the microneedles are arranged in a 10 × 10 array with a tip-to-tip spacing of 900μm.

[0019] 4) Add 150 μL of HMHA hydrogel containing 10 mg / mL CyOH & CO Nps and centrifuge to fill the microneedle tip voids in the mold; then scrape off the hydrogel from the base portion and dry in a ventilated area for 1 hour. Repeat this step three times to fill the needle tip cavities; finally, pour 150 μL of LMHA hydrogel containing 10 mg / mL CyOH & CO Nps onto the surface of the micromold and centrifuge to form the base layer. After drying overnight at room temperature, carefully peel the HA-encapsulated CyOH & CO Nps microneedles from the mold with adhesive tape to obtain a microneedle patch that uses cyanin-derived pH-responsive photofunctional molecules to inhibit bacterial infection of wounds.

[0020] The beneficial effects of this invention are:

[0021] This invention proposes a microneedle patch that seamlessly integrates fluorescence imaging and photodynamic therapy activated by infection microenvironment with synergistic antibacterial activity of carbon monoxide (PDT-CO) gas to inhibit bacterial infection in wounds using anthocyanin-derived pH-responsive photofunctional molecules. Our approach begins with the design and synthesis of a high-performance phototherapy agent based on anthocyanin derivatives. As a marker of bacterial metabolism, many bacteria produce organic acids as metabolic byproducts, leading to a decrease in the pH of their surrounding environment. The probe developed in this invention can sense and dynamically adjust its fluorescence and PDT properties in response to changes in acidity in the infection microenvironment. This infection-activated photosensitizer not only facilitates precise infection monitoring but also enables efficient, site-specific antibacterial therapy. Subsequently, we co-assembled the hydrophobic probe with the reactive oxygen species (ROS)-reactive CO donor CORM-401 to form a biocompatible therapeutic nanosystem, which was further encapsulated in a hyaluronic acid (HA) microneedle patch to enhance its mechanical penetration and controlled-release activity at the infection site. At the site of infection, bacteria secrete various extracellular enzymes, including hyaluronidase, which can degrade the microneedle matrix, thereby triggering the release of nanomedicines. When encountering acid-producing bacteria, the nanoreagent undergoes molecular structural changes, producing a bright fluorescent signal output. This allows for real-time, sensitive detection of infection and differentiation of infection severity, providing a basis for treatment decisions. Simultaneously, activating the PDT properties of the nanoprobes not only directly kills bacteria through oxidative damage but also triggers CORM-401 to release CO for adjuvant gas therapy. Therefore, the combination of PDT and CO gas cascades produces a powerful bacterial elimination effect, significantly inhibiting bacterial biofilm-mediated infections. This customized therapeutic microneedle platform ingeniously combines infection-activated bioimaging with synergistic antimicrobial therapy, offering enormous potential for effective and personalized management of infection-related diseases. Attached Figure Description

[0022] Figure 1 The present invention provides a 1H NMR spectrum of CyOH alkaline environment. 1 H NMR)

[0023] Figure 2 The present invention provides a hydrogen nuclear magnetic resonance spectrum of CyOH in an acidic environment. 1 H NMR)

[0024] Figure 3 The present invention provides a carbon NMR spectrum of the CyOH acidic environment. 13 (C NMR)

[0025] Figure 4 The UV absorption of CyOH varies in different pH buffer solutions.

[0026] Figure 5 The pKa is calculated based on the UV absorption at the maximum absorption peak of 550 nm at different pH values ​​using the following formula:

[0027] log[(Amax–A) / (A–Amin)]=pH–pKa

[0028] Figure 6 The fluorescence emission changes of CyOH in different pH buffer solutions were shown, with an excitation wavelength of 550 nm.

[0029] Figure 7 1,3-Diphenylisobenzofuran (DPBF) reacts with CyOH at 30 mW / cm under acidic pH conditions. -2 Changes in ultraviolet absorption under white light irradiation.

[0030] Figure 8 The UV absorption changes were defined for a mixture of DPBF and CyOH exposed to light in an alkaline pH environment, a mixture of DPBF and CyOH not exposed to light in an acidic pH environment, and a DPBF solution exposed to light only.

[0031] Figure 9 DLS data and TEM images of CyOH nanoparticles.

[0032] Figure 10 DPBF reacts with CyOH & CO under acidic pH conditions at 30 mW / cm². -2 Changes in ultraviolet absorption under white light irradiation.

[0033] Figure 11 The 1H NMR spectrum of the CO detection probe.

[0034] Figure 12 The fluorescence emission variables at 515 nm are the changes of CO detection probe and CyOH & CO Nps under acidic conditions, under alkaline conditions, with CO detection probe alone under white light irradiation time, and with CO detection probe and CyOH & CO Nps under acidic conditions without white light irradiation over time.

[0035] Figure 13 This is a quantitative graph showing the detection of cell viability using the CCK8 assay at different concentrations of nanoparticles under light and dark conditions.

[0036] Figure 14 Representative images and quantitative graphs of bacterial growth inhibition by different nanoparticles under light and dark conditions using plate counting method.

[0037] Figure 15 Optical and scanning electron microscope images of CyOH & CO microneedles.

[0038] Figure 16 To quantify the fluorescence signal and fluorescence intensity emitted by the MN patch for small animal in vivo imaging (IVIS).

[0039] Figure 17 Digital photographs of the lesion sites and quantitative graphs of the degree of healing were taken from different treatment groups over seven days.

[0040] Figure 18 Weight changes in different treatment groups over seven days.

[0041] Figure 19 HE staining images of vital organs (heart, liver, spleen, lung, and kidney) in different treatment groups.

[0042] Figure 20 Complete blood count and blood biochemistry analysis were performed on the final treatment group and healthy mice. Detailed Implementation

[0043] The technical solution of the present invention will be further explained and described below with reference to specific embodiments.

[0044] This embodiment describes a method for preparing a microneedle patch that utilizes cyanin-derived pH-responsive photofunctional molecules to inhibit bacterial infection in wounds. The specific steps are as follows:

[0045] Example 1 [Preparation of Cyanide-Derived Photofunctional Molecules]

[0046] Synthesis route:

[0047]

[0048] 1) Dissolve 1.5 mL of 2,3,3-trimethylindole in 17 mL of acetonitrile, remove oxygen three times, add 1.0 g of 3-iodo-1-propanol to the above system, and heat under reflux for 24 h under nitrogen protection; after the reaction system is cooled to room temperature, remove the solvent by vacuum distillation; then wash the product with petroleum ether, remove the petroleum ether by rotary evaporation, dissolve it in a small amount of acetonitrile, pour it into cooled diethyl ether, precipitate the precipitate and filter to obtain 1-(3-hydroxypropyl)-2,3,3-trimethyl-3H-indole;

[0049] 2) Subsequently, the above product was dissolved in 15 mL of acetonitrile solution with 4-[bis(4-methoxyphenyl)amino]benzaldehyde. Piperidine was added dropwise, and the reaction system was heated to 80 °C and reacted under nitrogen protection for 10 h. The purified product was denoted as CyOH.

[0050] The structure of the polymer was characterized using 1H NMR, 13C NMR, and UV-Vis absorption spectroscopy. The molecular structures in acidic and alkaline environments are as follows:

[0051]

[0052] like Figure 1 The 1H NMR spectrum under alkaline conditions shown is as follows:

[0053] 1 H NMR(400MHz, DMSO-d6)δ7.36(d,J=8.3Hz,2H),7.21–6.81(m,10H),6.76–6.50(m,5H),6.05(d,J=16.4Hz ,1H),3.88(t,J=12.1Hz,2H),3.71(s,6H),3.57–3.21(m,2H),1.68(d,J=13.3Hz,2H),1.37–0.84(m,6H).

[0054] like Figure 2 The 1H NMR spectrum under acidic conditions shown is as follows:

[0055] 1 H NMR(400MHz,DMSO-d6)δ8.36(d,J=15.8Hz,1H),8.06–7.92(m,2H),7.88–7.7 3(m,2H),7.55(ddd,J=13.4,7.5,1.2Hz,2H),7.40(d,J=15.9Hz,1H),7.28–7 .14(m,4H),7.08–6.98(m,4H),6.78–6.67(m,2H),4.83(s,1H),4.58(t,J=7. 0Hz, 2H), 3.78 (s, 6H), 3.52 (t, J = 5.7Hz, 2H), 2.08–1.93 (m, 2H), 1.75 (s, 6H).

[0056] like Figure 3 The carbon NMR spectrum under acidic conditions shown is as follows:

[0057] 13 C NMR(101MHz,DMSO-d6)δ180.90,157.98,143.59,141.51,138.07,133.71,129.36,1 28.77,125.59,123.36,115.83,114.63,107.77,57.94,55.89,55.40,51.88,26.58.

[0058] Verification of the pH responsiveness and photodynamic properties of CyOH:

[0059] pH titration: Buffer solutions with different pH values ​​were prepared using an INESA PHS-2F pH meter. The pH meter was calibrated at 25°C using standard buffer solutions with pH values ​​of 4.01±0.01, 6.18±0.01, and 9.00±0.01. Phosphate buffer solutions with different pH values ​​were prepared for pH titration. All aqueous buffer solutions were freshly prepared and stored in a refrigerator (for use within one week). A concentrated stock solution of 50 μM CyOH was prepared in DMSO. The stock solution was quantitatively added to buffer solutions of different pH values ​​to obtain a final probe concentration of 2.5 μM. The solution was thoroughly stirred before spectral measurement. The absorbance change with pH titration was recorded using a UV spectrophotometer. Figure 4 At acidic pH, a new absorption peak appears at 550 nm due to the formation of a highly conjugated open structure, while the intensity decreases at 342 nm. Notably, when the pH decreases from 7.5 to 5, the absorbance of the CyOH molecule at 550 nm increases rapidly, consistent with the observed color shift. pH titrations were then performed in buffer solutions with different pH values, and the pKa value was calculated to be approximately 6.39 based on S-shaped curve fitting of the absorbance at 550 nm at different pH values. Figure 5 ).

[0060] Given that fluorescence imaging provides real-time feedback and high sensitivity, we subsequently monitored the fluorescence spectra of CyOH at different pH levels to assess its potential for fluorescence-sensing infection. Figure 6 As shown, the near-infrared emission band [λem 750 nm, λex 550 nm] increases with decreasing pH. Due to the on / off activation of near-infrared fluorescence, the fluorescence signal at 750 nm increases significantly by 6-fold from pH 8 to 4.0.

[0061] CyOH photodynamic performance testing:

[0062] Using 1,3-diphenylisobenzofuran (DPBF) as a sensitive ROS indicator, the ability of CyOH to generate ROS under different pH conditions was investigated in detail. DPBF can effectively capture and interact with ROS, resulting in a significant decrease in absorbance at 412 nm.

[0063] DPBF was dissolved in DMSO and added to PBS (pH=5) containing CyOH-Nps (50 μg / mL), resulting in a DPBF concentration of approximately 100 μM / mL. The UV absorbance at this point was measured using a UV spectrophotometer and recorded as 0. Subsequently, the sample was illuminated with a white light flashlight (using a filter to remove absorbable light from DPBF and reduce the influence of photobleaching on the experiment), and the UV absorbance was measured every minute. Similarly, measurements were performed on the CyOH-Nps group without light, the group containing only DPBF with light, and the CyOH-Nps+DPBF alkaline group, and the data were recorded. Figure 7 , 8 Under acidic pH conditions, in the presence of CyOH and under the same white light irradiation, the absorbance of DPBF at the 412 nm absorption peak decreased sharply. Furthermore, the decrease in DPBF absorbance became more pronounced with prolonged white light irradiation time. This phenomenon strongly suggests that DPBF is catalytically oxidized and converted by ROS during photosensitization. As a control, in an acidic pH environment, neither the mixture of DPBF and CyOH exposed to light nor the DPBF solution exposed only to light showed a significant reduction in absorbance. This observation highlights that both photoactivated and acid-activated CyOH are essential for triggering ROS generation.

[0064] Example 2 [Preparation of CyOH Nanoparticles]

[0065] Add 8.0 mg CyOH, 40 mg DSPE-PEG, and 10 mL of water to 1.0 mL DMSO. Stir rapidly (1400 rpm). Insert the needle below the liquid surface and add the solution dropwise over approximately five minutes. Dialyze for 4 hours, changing the water every half hour.

[0066] The size and morphology of the obtained NPs were characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM). Figure 9 As shown, DLS data revealed an average diameter of 126 nm for CyOH NPs and a polydispersity index (PDI) of 0.15. Simultaneously, TEM imaging confirmed the uniform spherical structure of the CyOH NPs, with an average diameter of 110 nm. The difference in diameter between the DLS and TEM measurements can be attributed to the drying and shrinkage of the NPs during TEM sample preparation.

[0067] Example 3 [Preparation of CORM-401 containing CyOH and CO prodrug]

[0068] To achieve a synergistic antibacterial effect through the combination of photodynamic therapy (PDT) and CO gas therapy, we further loaded the ROS-responsive CO-releasing precursor CORM401 into CyOH NPs. This was accomplished by co-assembling CyOH and CORM-401 with DSPE-PEG2000, thereby generating CyOH&CO NPs. The procedure is as follows:

[0069] Add 8.0 mg CyOH, 2.0 mg CORM-401, 40 mg DSPE-PEG, and 10 mL of water to 1.0 mL DMSO. Stir rapidly (1400 rpm). Insert the needle below the liquid surface and slowly add the solution dropwise over approximately five minutes. Dialyze for 4 hours, changing the water every half hour.

[0070] When CyOH & CO Nps encounter an acidic infection microenvironment, they initiate a two-pronged antibacterial action. First, CyOH & CO Nps act as a highly efficient photosensitizer, generating cytotoxic ROS to damage bacteria. Subsequently, the accumulation of local ROS triggers the controlled release of CO from CyOH & CO Nps, inhibiting bacterial growth by disrupting the plasma membrane and inducing metabolic disturbances.

[0071] CyOH&CO Nps Verification of CO Gas Release:

[0072] It has been reported that CORM-401 can rapidly release CO under ROS-induced strong oxidative stress. Therefore, we expect a series of events involving photoinduced ROS generation and ROS-activated CO dissociation in CyOH & CO Nps under white light irradiation. To verify our hypothesis, we first used DPBF as a ROS indicator to evaluate the ROS generation capacity of CyOH & CO Nps. Under acidic conditions, the absorbance of the CyOH & CO Nps and DPBF mixture decreased with prolonged exposure to white light, possibly due to the absorption peak at 412 nm being absorbed by the emerging ROS-activated CO Nps. 1 O2 oxidation ( Figure 10 This result indicates that incorporating CyOH into CyOH-coNPs does not affect their acid reactivity and ROS generation performance. To quantitatively assess the amount of CO generated during light irradiation, a CO detection fluorescent probe was synthesized based on existing methods, and its performance was measured using 1H NMR spectroscopy. 1 The structure of the probe was characterized by H NMR. Figure 11 ).

[0073] 1H NMR (400MHz, Chloroform-d) δ8.52(s,1H),8.05–7.70(m,1H),7.66–6.99(m,7H),6.67–6.32(m,6H),3.31(q,J=7.1Hz,8H),1.15(t,J=7.0Hz,12H).

[0074] After reacting with CO, the fluorescence intensity of the probe at the characteristic peak of 515 nm increased. For example... Figure 12 As shown, in the presence of CyOH & CO Nps, prolonged light irradiation time significantly increased the concentration of dissolved CO, while CO release without light irradiation was negligible. As a control, only CORM-401 remained relatively stable under light irradiation and did not release CO.

[0075] After confirming the PDT effect and cascade activation of CO gas release by CyOH&CO Nps, we comprehensively evaluated its antibacterial efficacy. Biocompatibility is a fundamental requirement in the development of safe and effective bactericidal nanoparticles. Therefore, we first assessed the cytotoxicity of CyOH&CO Nps against L929 mouse fibroblasts using the Standard Cell Counting Kit-8 (CCK-8) assay. Notably, no significant cytotoxicity was observed after 24 hours of exposure to CyOH&CO Nps at concentrations ranging from 0 to 200 μg mL⁻¹. Figure 13 In particular, even at a high concentration of NP (200 μg mL⁻¹), cell viability exceeded the 85% threshold. The low cytotoxicity of CyOH&CO NPs to normal mammalian cells is primarily attributed to their minimal dark toxicity. Interestingly, even under light irradiation, cells maintained a high viability of over 80%. This observation can be explained by the weak PDT effect of CyOH&CO Nps under neutral conditions, further highlighting the high biocompatibility of pH-activated nanoparticles.

[0076] We evaluated the antimicrobial efficacy of CyOH&CO Nps against *Escherichia coli* (Gram-negative) and *Staphylococcus aureus* (Gram-positive) using a standard plate count method. After various treatments, bacteria cultured in lysogeny broth (LB) were transferred to LB agar (LBA) plates. After overnight incubation, we determined the antimicrobial effect by quantifying colony-forming units (CFU). Figure 14 As shown, compared to the control group treated with PBS alone, we observed only a slight reduction in the number of bacteria incubated with CyOH & CO Nps in the absence of light irradiation, with a CFU decrease of only 8.1%. This result indicates that CyOH & CO Nps have minimal dark toxicity to bacterial cells. Similarly, CORM-401 under light irradiation exhibited limited antibacterial activity due to the lack of ROS-activated CO release. Notably, "CyOH NPs + light" treatment induced moderate levels of bacterial inhibition, with a 65.3% reduction in CFU due to photosensitization-induced ROS-mediated cytotoxicity. Most significantly, the survival rates of *Escherichia coli* and *Staphylococcus aureus* were significantly reduced when CyOH & CO Nps were combined with light irradiation.

[0077] Example 4 [Preparation of Microneedle Patches]

[0078] To enhance the mechanical penetration of nanomedicines into complex infection systems, such as biofilm infections and deep skin infections, we further incorporated CyOH&CO Nps into microneedle (MN) patches to develop a transdermal diagnostic and therapeutic platform.

[0079] Preparation method of microneedle patches:

[0080] 1) A polydimethylsiloxane (PDMS) micromold with rectangular conical holes is created by pouring a premixed PDMS solution into a master mold, then degassing (in a vacuum oven for 15 minutes) and curing (at 65°C for 5 hours).

[0081] Each microneedle has a 500μm × 500μm square base that tapers to a height of 1000μm. The microneedles are arranged in a 10×10 array with a tip-to-tip spacing of 900μm.

[0082] 2) Add 150 μL of HMHA hydrogel (500 kDa) containing CyOH & CO Nps (10 mg / mL) and fill the voids by centrifugation (3000 g, 5 min). Then scrape off the HA hydrogel from the base portion and dry in a ventilated area for 1 hour. Repeat this step three times to fill the needle tip cavity. Finally, pour 150 μL of LMHA hydrogel (10 kDa) containing CyOH & CO Nps (10 mg / mL) onto the micromold surface and centrifuge (1000 g, 10 min) to form the base layer. After drying overnight at room temperature, carefully peel the HA-encapsulated CyOH & CO Nps microneedles from the mold using adhesive tape; obtain the CyOH-CO@MN patch.

[0083] Optical images of microneedle patches ( Figure 15 The image shows that the microneedles are arranged in a 10×10 array, which encapsulates CyOH & CONps.

[0084] We used a mouse wound infection model to evaluate its ability to monitor pH changes in real time under in vivo infection conditions. First, we carefully created an 8 mm diameter circular wound on the dorsal skin of BALB / c mice and then inoculated it with a Staphylococcus aureus suspension to induce infection. CyOH-CO@MN patches were implanted into the infected wound at time intervals of 2, 6, 12, and 24 hours post-inoculation to demonstrate its ability to monitor the infection microenvironment in situ. The MN patch was gently pressed onto the wound for 3 minutes, and the substrate was removed after 0.5 hours, leaving the needle in the dermis. The fluorescence signal emitted by the MN patch was continuously monitored using IVIS. Figure 16 As shown, the uninoculated control wound exhibited minimal near-infrared radiation during the 12-hour observation period. Notably, CyOH-CO@MN showed a recognizable red fluorescence signal as early as 2 hours after bacterial inoculation. The fluorescence intensity gradually increased as the infection progressed, peaking at 12 hours post-infection. These results demonstrate that CyOH-CO@MN can be successfully used for the accurate diagnosis of infection, even in its early stages.

[0085] The antibacterial and wound-healing capabilities of CyOH-CO@MN were then evaluated in a mouse subcutaneous infection model. A Staphylococcus aureus suspension was injected into a carefully prepared wound on the dorsal skin of mice to establish infection. Six hours after wound induction, infected mice were randomly divided into six groups of three mice each, receiving different treatments: (1) PBS, (2) single-color light, (3) CyOH@MN + light, (4) CyOH-CO + light, (5) CyOH-CO@MN, and (6) CyOH-CO@MN + light. In cases involving light exposure, after administration of the appropriate formulation, the mouse wounds were exposed to white light at an intensity of 0.2 W / cm² for 10 minutes.

[0086] Throughout the treatment process, digital photographs of the lesion site were taken to dynamically monitor the wound healing progress in each group. For example... Figure 17 As shown, on day 3 post-treatment, wounds in the PBS group, light irradiation group, or CyOH-CO@MN group continued to show signs of infection, characterized by severe ulceration and swelling around the wound. In contrast, the groups receiving CyOH@MN + light irradiation or CyOH-CO@MN + light irradiation showed reduced ulceration and a clear trend towards wound healing. Seven days after treatment, wounds treated with PBS, light irradiation, or CyOH-CO@MN alone still showed significant yellow bacterial biofilm and obvious abscesses, indicating that these interventions had limited effectiveness in resolving wound infection. The wound area in the CyOH@MN + light irradiation group was significantly reduced. Based on the pseudo-color map of wound healing and quantitative wound area analysis, the wound healing rate was approximately 77%, a significant improvement compared to the control group (48.53±3.19%), the light irradiation group alone (58.36±3.06%), and the CyOH-CO@MN group (63.57±2.56%). Clearly, infected wounds treated with CyOH-CO@MN plus phototherapy healed almost completely, with only minimal scarring observed 7 days after treatment. The wound healing rate exceeded 87%. The effective antibacterial effect of PDT / CO gas therapy likely accelerated bacterial clearance and wound healing. Furthermore, it is worth emphasizing that CyOH-CO@MN + phototherapy exhibited better anti-infection effects compared to the free CyOH-CO@MN group, which is attributed to the microneedles promoting the penetration and retention of nanoparticles in the infected skin area.

[0087] Finally, the overall toxicity of CyOH-CO@MN was evaluated from multiple perspectives. For example... Figure 18 As shown, firstly, we noted that there was no significant fluctuation in the body weight of mice in all groups throughout the treatment period, indicating that CyOH-CO@MN had minimal adverse effects on mouse growth. Furthermore, histological sections of major organs (including heart, liver, spleen, lungs, and kidneys) were examined on day 7 after CyOH-CO@MN + light treatment. Figure 19Compared to healthy tissue, histological abnormalities and tissue damage were negligible. Blood samples were also collected from the mice at the end of treatment for routine blood analysis.

[0088] like Figure 20 As shown, complete blood count indicators, including white blood cell count (WBC), red blood cell count (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), and mean corpuscular hemoglobin concentration (MCHC), were all within their respective normal ranges (shown by dashed lines). Furthermore, liver and kidney function parameters were examined, indicating that the use of CyOH-CO@MN did not cause acute liver or kidney injury. In summary, these findings confirm the favorable biocompatibility of CyOHCO@MN for infection imaging and antimicrobial therapy.

[0089] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. For those skilled in the art, any changes or substitutions that can be easily conceived, such improvements and modifications, are also considered to be within the scope of protection of the present invention.

Claims

1. A pH-responsive photo-functional molecule derived from a cyanine, characterized in that, The structural formula of the molecule is: The structures of the molecule in acidic and basic environments are respectively:

2. A method for preparing a cyanin-derived pH-responsive photofunctional molecule as described in claim 1, characterized in that, The steps of the method are as follows: 1) 1.5 mL of 2,3,3-trimethylindole is dissolved in 17 mL of acetonitrile, and after being deoxygenated for 3 times, 1.0 g of 3-iodo-1-propanol is added to the above system, and heated to reflux under nitrogen protection for 24 h; after the reaction system is cooled to room temperature, the solvent is removed by reduced pressure distillation; then the product is washed with petroleum ether, and after removing the petroleum ether by rotary evaporation, it is dissolved in a small amount of acetonitrile, poured into cooled diethyl ether, precipitated and filtered to obtain 1-(3-hydroxypropyl)-2,3,3-trimethyl-3H-indole; 2) 1-(3-hydroxypropyl)-2,3,3-trimethyl-3H-indole and 4-[bis(4-methoxyphenyl)amino]benzaldehyde are dissolved in 15 mL of acetonitrile solution, and after adding piperidine dropwise, the reaction system is heated to 80℃, and reacted for 10 h under nitrogen protection; after purification, the product, i.e. the cyanine derivative pH-responsive optical functional molecule, is obtained.

3. A microneedle patch for inhibiting bacterial infection in a wound using the pH-responsive photo-functional molecule derived from the cyanine of claim 1. Each microneedle in the microneedle patch has a square base of 500 μm x 500 μm, each microneedle gradually tapers from the base to the tip, with a height of 1000 μm, and the microneedles are arranged in a 10 x 10 array with a tip-to-tip spacing of 900 μm; The tip is molded by HMHA hydrogel containing CyOH&CO Nps with a concentration of 10 mg / mL, and the base is molded by LMHA hydrogel containing CyOH&CO Nps with a concentration of 10 mg / mL; the HMHA hydrogel is 500 KDa, and the LMHA hydrogel is 10 KDa; The preparation method of CyOH&CO Nps is: 8.0 mg of the cyanine derivative pH-responsive optical functional molecule, 2.0 mg of CORM-401, 40 mg of DSPE-PEG and 10 mL of water are added to 1.0 mL of DMSO, and the solution is stirred rapidly at a speed of 1400 r / min, a needle is inserted below the liquid surface, and the solution is added slowly dropwise for five minutes; dialysis is performed for 4 h, and the water is changed every half hour to obtain CyOH&CO NPs.

4. A preparation method of the microneedle patch for inhibiting bacterial infection of a wound by using the cyanine derivative pH-responsive optical functional molecule according to claim 3, and the specific steps are as follows: 1) 1.5 mL of 2,3,3-trimethylindole is dissolved in 17 mL of acetonitrile, and after being deoxygenated for 3 times, 1.0 g of 3-iodo-1-propanol is added to the above system, and heated to reflux under nitrogen protection for 24 h; after the reaction system is cooled to room temperature, the solvent is removed by reduced pressure distillation; then the product is washed with petroleum ether, and after removing the petroleum ether by rotary evaporation, it is dissolved in a small amount of acetonitrile, poured into cooled diethyl ether, precipitated and filtered to obtain 1-(3-hydroxypropyl)-2,3,3-trimethyl-3H-indole; CyOH was prepared by mixing 1-(3-hydroxypropyl)-2,3,3-trimethyl-3H-indole and 4-[bis(4-methoxyphenyl)amino]benzaldehyde in 15 mL acetonitrile, then adding piperidine drop by drop, and heating the reaction system to 80°C under nitrogen protection for 10 h; the product was obtained after purification and was recorded as CyOH; 2) 8.0 mg of CyOH, 2.0 mg of CORM-401, 40 mg of DSPE-PEG, 10 mL of water were added in 1.0 mL of DMSO, and the mixture was stirred at 1400 r / min, the needle was inserted below the liquid surface, and the mixture was slowly added dropwise for five minutes; the mixture was dialyzed for 4 h, and the water was changed every half hour to obtain CyOH&CONPs; 3) A polydimethylsiloxane (PDMS) micromold with rectangular tapered holes was created by pouring a premixed PDMS solution into a master mold, then degassing and curing; the design of the mold followed the following rules: each microneedle had a square base of 500 μm x 500 μm, each microneedle gradually tapered from the base to the tip, the height was 1000 μm, the microneedles were arranged in a 10 x 10 array, and the tip-to-tip distance was 900 μm; 4) 150 μL of HMHA hydrogel containing CyOH&CONPs at a concentration of 10 mg / mL was added, and the microneedle tip cavity was filled by centrifugation; then the hydrogel on the base part was scraped off, and dried in a ventilated place for 1 h; this step was repeated three times to fill the needle tip cavity; finally, 150 μL of LMHA hydrogel containing CyOH&CONPs at a concentration of 10 mg / mL was poured onto the surface of the micromold and centrifuged to form a base layer; after drying overnight at room temperature, the HA-encapsulated CyOH&CONPs microneedle patch was carefully peeled off from the mold, and a microneedle patch for inhibiting bacterial infection in wounds using a pH-responsive photosensitive molecule derived from a cyanine was obtained.

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