Intelligent antibacterial hydrogel of photosensitizer combined with antibiotics and preparation method and application thereof

Abstract

The application discloses an intelligent antibacterial hydrogel combined with a photosensitizer and an antibiotic as well as a preparation method and application thereof. The hydrogel takes oxidized dextran 40 as a hydrogel matrix, bonds 5-amino hexyl acetylpropionate into OAHAL through a Schiff base reaction, and is then crosslinked with amikacin to be prepared. The hydrogel has pH sensitivity, can effectively release drugs under an inflammatory slightly acidic environment, realizes on-demand release of the drugs, avoids overuse of the antibiotic, reduces toxic side effects, simultaneously loads two kinds of bactericidal drugs, 5-amino hexyl acetylpropionate and amikacin, and synergistically resists bacteria through two different mechanisms of photodynamic therapy and antibiotics, so that the antibacterial effect is significantly improved, and bacterial colonies caused by various types of wounds can be eliminated, and bacterial infection of a wound surface can be effectively prevented or treated.

Description

Technical Field

[0001] This invention belongs to the fields of nanomedicine formulation and polymer chemistry, specifically relating to a photosensitizer combined with an antibiotic intelligent antibacterial hydrogel, its preparation method, and its application. Background Technology

[0002] Skin trauma is a widespread and easily occurring global health problem. If not treated in time, it can lead to more serious bacterial infections, making the wound more difficult to heal. The treatment of wound infections is still mainly based on traditional therapies such as debridement and irrigation, wound dressing, and drug therapy. Because antibiotics can kill bacteria quickly and efficiently, they are still the first choice for treating bacterial infections. However, the use of antibiotics still faces several major challenges: (1) Low-dose antibiotics have poor sterilization effects, while high-dose antibiotics can cause various toxic side effects such as nephrotoxicity and ototoxicity; (2) The bacterial colonies and microbial environments of wounds caused by different types of skin trauma are different. For example, most bacterial infections in acute trauma are caused by Gram-positive bacteria, including Staphylococcus aureus and Streptococcus; Gram-negative bacteria account for a higher proportion in chronic trauma bacterial infections, including Pseudomonas aeruginosa, Escherichia coli, and Candida; therefore, a single antibiotic is difficult to effectively kill all the bacterial colonies in the wound; (3) Overuse of antibiotics can lead to bacterial resistance problems, such as the emergence of superbugs. Therefore, achieving on-demand release of antibiotics and finding new antibacterial drugs are currently major focuses in solving bacterial infection problems.

[0003] Amikacin (AMK) is an aminoglycoside antibiotic. Its mechanism of action primarily involves binding to the 30S subunit of bacterial ribosomes, inhibiting bacterial protein synthesis, disrupting the integrity of the bacterial cell wall, and thus leading to bacterial cell membrane destruction and death. Amikacin has an antibacterial spectrum against a wide range of Gram-negative bacteria and a small number of Gram-positive bacteria, primarily including *Pseudomonas aeruginosa*, *Escherichia coli*, *Proteus*, *Klebsiella*, *Serratia marcescens*, and penicillin-resistant *Staphylococcus aureus*. However, it is not very effective against most Gram-positive bacteria. Clinically, amikacin is often used to treat site infections caused by these susceptible bacteria, including bronchitis, pneumonia, bacterial endocarditis, cholecystitis, cholangitis, osteomyelitis, abdominal infections, urinary tract infections, and skin and soft tissue infections. However, the use of amikacin may cause ototoxicity and nephrotoxicity, which limits its clinical application.

[0004] 5-Aminolevulinic acid (5-ALA), also known as 5-aminolevulinic acid, is an intermediate product in the synthesis of heme in mammalian cells. While it lacks photosensitizing properties, it can synthesize an endogenous photosensitizer molecule, protoporphyrin (PpIX), within the mitochondrial inner membrane. When exogenously administered, 5-ALA specifically enters high-metabolic tissues or organisms (such as tumor cells, macrophages, and bacteria), forming the photosensitizer PpIX. Under light conditions, PpIX generates reactive oxygen species (ROS), thereby inducing apoptosis in cells or bacteria. However, due to its hydrophilic and zwitterionic properties, 5-ALA has a weak ability to cross biomembrane barriers and struggles to penetrate bacterial biofilms. Studies have shown that hexyl 5-aminolevulinate (5-ALA·HAL), with its higher lipophilicity, more easily penetrates bacterial barriers and reaches the cytoplasm, exhibiting strong cytotoxic effects against most Gram-positive and Gram-negative bacteria. Therefore, 5-ALA·HAL, used in photodynamic therapy (PDT), holds promise as a novel antibacterial agent and can effectively alleviate problems such as antibiotic resistance. However, studies have found that 5-ALA·HAL degrades much faster than 5-ALA under physiological conditions, requiring prolonged and repeated administration in clinical practice, which increases the risk of harm to normal physiological cells. Therefore, there is an urgent need to design a 5-ALA·HAL prodrug that is stable under normal conditions but active in the wound microenvironment, releasing 5-ALA·HAL for specific antibacterial effects.

[0005] Simple drug layering used in clinical practice cannot alter the inherent pharmacokinetic characteristics and tissue distribution properties of drugs, while the efficacy and toxicity of drugs are closely related to their absorption, distribution, and uptake in vivo. Loading combination drugs onto the same carrier for simultaneous targeted delivery to the lesion site, and inhibiting or blocking cell proliferation through multiple mechanisms at the optimal dose ratio, holds promise for achieving optimal synergistic effects with specific drug combinations, while simultaneously improving drug efficacy and effectively reducing toxicity. Therefore, there is an urgent need for a simple method to prepare a combined drug delivery system for the combined administration of amikacin and hexyl 5-aminolevulinate, achieving highly efficient bactericidal action and on-demand dosing, reducing drug toxicity, and helping to address the problem of bacterial resistance. Summary of the Invention

[0006] The purpose of this invention is to provide a photosensitizer combined with an antibiotic intelligent antibacterial hydrogel, its preparation method and application. This hydrogel has pH-sensitive properties, which can realize the on-demand release of antibiotics in the slightly acidic environment of inflammation, avoid the overuse of antibiotics, reduce the dosage and toxic side effects of antibiotics, and at the same time, the two drugs work together to kill bacteria through different mechanisms, which increases the antibacterial effect and can effectively prevent or treat bacterial infections of wounds.

[0007] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0008] A photosensitizer combined with an antibiotic intelligent antibacterial hydrogel is provided, which is prepared by using oxidized dextran 40 (ODex) as the hydrogel matrix, bonding 5-aminolevulinic acid hexyl ester with Schiff base reaction to obtain OAHAL; and then crosslinking it with amikacin.

[0009] According to the above scheme, the oxidized dextran 40 is prepared by oxidizing dextran 40 with sodium periodate, wherein the mass ratio of dextran 40 to sodium periodate is 1.5~1:1. Preferably, the molecular weight of dextran 40 is 40-50 kDa.

[0010] According to the above scheme, the mass ratio of oxidized dextran 40 to 5-aminolevulinic acid hexyl ester is 2.5~5:1.

[0011] According to the above scheme, the mass ratio of OAHAL to amikacin is 0.85~2.13:1, preferably 1~1.6:1, and more preferably 1.3-1.5:1.

[0012] A method for preparing the above-mentioned photosensitizer combined with antibiotic intelligent antibacterial hydrogel is provided, mainly including the following steps:

[0013] 1) Oxygenated dextran 40 (ODex) and the photosensitizer precursor 5-aminolevulinic acid hexyl ester hydrochloride (5-ALA·HAL) were mixed and subjected to a Schiff base reaction. The product OAHAL was then obtained by pH-sensitive imine bond coupling with pH-sensitive imine bond and followed by dialysis and lyophilization. The reaction formula is as follows:

[0014]

[0015] 2) The product OAHAL obtained in step 1) (denoted as product A) is cross-linked with amikacin (AMK). After dialysis and lyophilization, the dual-drug antibacterial hydrogel (ODAA) is obtained. The reaction formula is as follows:

[0016]

[0017] According to the above scheme, in step 1), the preparation of oxidized dextran 40 includes the following steps:

[0018] A certain amount of dextran 40 and sodium periodate were dissolved separately in deionized water. The sodium periodate solution was added dropwise to the dextran solution under stirring. The reaction was carried out in the dark and stirred for a period of time. After the reaction was terminated, stirring was continued in the dark. Finally, the product was obtained by dialyzing and lyophilization. The reaction formula is as follows:

[0019]

[0020] Preferably, the molecular weight of dextran 40 is 40-50 kDa.

[0021] Preferably, the mass ratio of dextran 40 to sodium periodate is 1.5 to 1:1.

[0022] Preferably, the reaction time is 12-24 hours, and after the reaction is terminated, stirring is continued in the dark for 2-3 hours.

[0023] Preferably, the reaction product is dialyzed with deionized water for 36-48 hours and lyophilized for 36-48 hours to obtain the oxidation product ODex.

[0024] Preferably, the reaction is terminated by adding ethylene glycol.

[0025] According to the above scheme, in step 1), the mass ratio of ODex to 5-aminolevulinic acid hexyl ester is 2.5~5:1.

[0026] According to the above scheme, in step 1), the pH of the reaction system is adjusted to 7-7.4 to carry out the Schiff base reaction. Preferably, the reagent used to adjust the pH is triethylamine.

[0027] According to the above scheme, in step 1), the reaction temperature is 40-60℃ and the reaction time is 3-4h.

[0028] According to the above scheme, in step 1), after the reaction is completed, the product OAHAL is obtained by dialysis with deionized water for 36-48 hours and freeze-drying for 36-48 hours.

[0029] According to the above scheme, in step 1), the solvent for the Schiff base reaction is deionized water.

[0030] According to the above scheme, in step 2), the mass ratio of OAHAL to amikacin is 0.85~2.13:1; preferably 1~1.6:1, more preferably 1.3-1.5:1.

[0031] According to the above scheme, in step 2), the crosslinking reaction time is 20s-10min; the temperature is 36-37.5℃.

[0032] According to the above scheme, in step 2), OAHAL and amikacin are dissolved in deionized water and then mixed to carry out a cross-linking reaction.

[0033] The present invention provides the application of the above-mentioned dual-drug combined intelligent antibacterial hydrogel in the preparation of antibacterial drugs, wherein the antibacterial drugs are simultaneously loaded with the photosensitizer 5-aminolevulinic acid hexyl ester and the antibiotic amikacin.

[0034] This invention utilizes dextran 40, which is non-toxic, harmless, biocompatible, inexpensive, and readily available, as a carrier. It employs a sodium periodate oxidation method to oxidize dextran into aldehyde-modified dextran, which is then bonded to the photosensitizer 5-aminolevulinic acid hexyl ester via a Schiff base reaction. Finally, the aldehyde group is cross-linked with the antibiotic amikacin, which has a polyamino structure, to prepare a pH-sensitive antibacterial hydrogel. This hydrogel has broad application prospects in the fields of antibacterial treatment and burn infection therapy.

[0035] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0036] 1. This invention provides a photosensitizer-integrated antibiotic intelligent antibacterial hydrogel, using oxidized dextran 40 as the hydrogel matrix, and simultaneously loading the photosensitizer precursor 5-aminolevulinic acid hexyl ester and the antibiotic amikacin. This hydrogel achieves excellent antibacterial effects through the combined action of photodynamic therapy and antibiotics, reducing the dosage of amikacin and thus lowering the hydrogel's toxic side effects. Simultaneously, it effectively improves the stability of 5-aminolevulinic acid hexyl ester, fully leveraging the antibacterial properties of the photosensitizer precursor. Furthermore, the resulting hydrogel is pH sensitive, enabling on-demand drug release in a slightly acidic environment during wound healing, avoiding antibiotic overuse and alleviating antibiotic abuse. The hydrogel's antibacterial spectrum includes most Gram-positive and Gram-negative bacteria, capable of eliminating bacterial colonies caused by various types of wounds, effectively preventing or treating bacterial infections of wounds, and has broad application prospects.

[0037] 2. The appropriate concentration of amikacin in the hydrogel obtained by this invention is beneficial to obtaining a uniformly distributed void structure, wherein the void structure can surround a large number of water molecules, maintain a moist environment for the wound surface, and help the wound heal. The hydrogel has good continuity, stable structure, and is easy to preserve.

[0038] 3. The hydrogel obtained by this invention can be used for injection or applied topically to a small area of ​​the infected site. It is convenient to use and has excellent antibacterial effect.

[0039] 4. This invention provides a method for preparing a photosensitizer combined with an antibiotic intelligent antibacterial hydrogel. It selects inexpensive, readily available, non-toxic, harmless, and biocompatible oxidized dextran 40 as the hydrogel matrix, uses Schiff base reaction to bond 5-aminolevulinic acid hexyl ester, and uses amikacin as both the antibiotic and cross-linking agent to obtain a dual-drug-loaded antibacterial hydrogel. The preparation process is simple and easy to control, the reaction conditions are mild, and the resulting hydrogel has a uniformly distributed porous structure with good continuity, structural stability, and is easy to store, exhibiting excellent antibacterial effects. Attached Figure Description

[0040] Figure 1The infrared spectra of the reaction raw material Dex, the intermediate product ODex, and OAHAL in Example 2 of this invention are shown.

[0041] Figure 2 For the reaction raw material Dex and intermediate product ODex in Example 2 of this invention 1 H NMR spectrum.

[0042] Figure 3 The reactant Dex, intermediate product ODex, and OAHAL in Example 2 of this invention 13 C NMR spectrum.

[0043] Figure 4 The UV radiation of the reaction raw material Dex, the intermediate product ODex, and OAHAL in Example 2 of this invention. Vis diagram.

[0044] Figure 5 The X-ray diffraction patterns of the reaction raw material Dex, the intermediate product ODex, and OAHAL in Example 2 of this invention.

[0045] Figure 6 The images shown are scanning electron microscope (SEM) images of the final product hydrogel in Example 2 of this invention, with the left image magnified 100 times and the right image magnified 1000 times.

[0046] Figure 7 These are the drug release curves of the hydrogels in different pH release media in each group of the embodiments of the present invention.

[0047] Figure 8 The results show the in vitro antibacterial properties of the hydrogels and control materials in the embodiments of the present invention against Staphylococcus aureus.

[0048] Figure 9 The results show the in vitro antibacterial properties of the hydrogels and control materials in the embodiments of the present invention against Pseudomonas aeruginosa. Detailed Implementation

[0049] To enable those skilled in the art to fully understand the technical solution and beneficial effects of the present invention, the following detailed description is provided in conjunction with specific embodiments.

[0050] Example 1

[0051] A method for preparing a photosensitizer-in-antibiotic smart antibacterial hydrogel is provided, comprising the following steps:

[0052] (1) Accurately weigh 5 g of dextran 40 (Dex, M w40 kDa was dissolved in 50 mL of deionized water at room temperature with stirring. Then, a NaIO4 solution (0.5 M, 107 mg / mL) was prepared and slowly added dropwise to 40 mL of the Dex solution using a dropper. The reaction was carried out at room temperature in the dark with stirring for 24 h. Then, 9 mL of ethylene glycol was added to terminate the reaction, and the reaction was continued for another 2 h under dark conditions with stirring. The mixture was then placed in a dialysis bag and dialyzed against deionized water (dialysis bag molecular weight cutoff: 1000 Da), with water changed every 6 h. After dialysis for 48 h, the sample was freeze-dried for 48 h to obtain the product oxidized dextran 40 (ODex).

[0053] The oxidation degree of the prepared ODex was determined by titration with hydroxylamine hydrochloride (NH2OH·HCl). The principle is as follows: the aldehyde group on ODex reacts with the active amino group on NH2OH·HCl to produce HCl. The amount and concentration of the aldehyde group in the system can then be calculated by titrating the HCl produced by the reaction with NaOH. The method is as follows: 50 mg of lyophilized ODex solid was accurately weighed and dissolved in 8 mL of NH2OH·HCl solution (0.25 M). The solution was left at room temperature for 3 h. Then, while stirring, a pre-prepared 0.1 M NaOH solution was added dropwise using a pH meter for titration. When the pH of the solution was greater than 4.4, the volume of NaOH solution used was recorded. A blank control was performed simultaneously with the sample test: 50 mg of ODex solid was weighed and dissolved in 8 mL of hydroxylamine hydrochloride solution (0.25 M), and the remaining experimental steps were the same as above.

[0054] The aldehyde content (n), i.e., the number of millimoles of aldehyde groups per gram of ODEX, is as follows:

[0055] Equation 2-1

[0056] The degree of oxidation of the aldehyde group (OX°), which is the number of units oxidized and opened out of 100 units of dextran, is as follows:

[0057]

[0058] Equation 2-2

[0059] In the formula, V1: the volume (mL) of NaOH (0.1 M) consumed by ODex in the sample solution.

[0060] V0: Blank solution is the volume (mL) of NaOH (0.1 M) consumed by Dex.

[0061] M: Molar concentration of NaOH (0.1 M) solution;

[0062] M W The weight of 40 units of dextran on the Dex chain is 160.

[0063] W: The weight (g) of the ODex weighed.

[0064] Calculated using the method described above, the oxidation degree of dextran 40 obtained in this embodiment is 32.8%.

[0065] (2) Weigh 500 mg of the prepared ODEX and dissolve it in 5 ml of deionized water. Stir slowly until completely defoamed. Accurately weigh 100 mg of 5-aminolevulinic acid hexyl ester hydrochloride (5-ALA·HAL) and dissolve it in deionized water. Add the prepared 5-ALA·HAL solution dropwise. Measure the pH of the mixed solution with a pH meter. Then slowly add triethylamine to adjust the pH of the mixed solution to 7.4. Transfer the mixed solution to a 25 ml flask and heat it in a water bath to 50 °C. After stirring for three hours, dialyze the reaction solution with deionized water (dialysis bag molecular weight cutoff = 1000 Da) to remove triethylamine and unreacted 5-ALA·HAL. Change the water every 4 h and dialyze for 24 h. Then freeze dry for 48 h to obtain ODEX / 5-ALA·HAL solid powder.

[0066] Subsequently, the loading and grafting rate of 5-aminolevulinic acid hexyl ester were determined. The specific method is as follows: 10 mg of OAHAL powder was weighed, and the mass fraction of nitrogen atoms (m) was determined by an elemental analyzer. N With C atomic mass fraction m C The mass fraction n of N atoms can be deduced. N The mole fraction n of C atoms C This allows for the calculation of drug loading (DLC) and grafting rate (GR).

[0067]

[0068]

[0069] In the formula, Mw 5-ALA.HAL The molecular weight of 5-aminolevulinic acid hexyl ester

[0070] Ar N The relative atomic mass of N atom

[0071] The drug loading of 5-aminolevulinic acid hexyl ester in OAHAL was determined to be 21.03% and the grafting rate was 27.93% using the above method.

[0072] (3) Weigh a certain amount of amikacin sulfate (AMK) sample and dissolve it in deionized water to prepare 58.5 mg / ml. Dissolve ODEX / 5-ALA·HAL in deionized water to prepare 100 mg / ml for later use. Mix the two solutions at a volume ratio of 500 ml: 400 ml with ODEX / 5-ALA·HAL:AMK, pour the mixture into a small glass slide, stir thoroughly, and then place it in a constant temperature shaking oven at 37°C for 8 minutes to form a hydrogel. After freeze-drying, the photosensitizer combined with antibiotic intelligent antibacterial hydrogel can be obtained.

[0073] Example 2

[0074] A method for preparing a photosensitizer-in-antibiotic smart antibacterial hydrogel is provided, comprising the following steps:

[0075] (1) Same as Example 1

[0076] (2) Same as Example 1

[0077] (3) Weigh a certain amount of amikacin sulfate (AMK) sample and dissolve it in deionized water to prepare 58.5 mg / ml. Dissolve ODEX / 5-ALA·HAL in deionized water to prepare 100 mg / ml for later use. Mix the two solutions at a volume ratio of 500 ml: 600 ml with ODEX / 5-ALA·HAL:AMK, pour the mixture into a small glass slide, stir thoroughly, and then place it in a constant temperature shaking oven at 37°C for 1 minute to form a hydrogel. After freeze-drying, the photosensitizer combined with antibiotic intelligent antibacterial hydrogel can be obtained.

[0078] To fully understand the various properties of the ODex / 5-ALA·HAL / AMK antibacterial hydrogel prepared in this embodiment, corresponding tests were conducted, including FTIR, 1 H NMR, 13 C NMR UV Vis, SEM, and in vitro drug release performance experiments are detailed below:

[0079] (1) Infrared characterization

[0080] Infrared spectroscopy analysis was performed on samples of the raw material Dex, intermediate product ODex, and ODex / 5-ALA·HAL from Example 2. The resulting spectra are shown below. Figure 1 As shown. The infrared spectrum of Dex is from left to right 3431 cm⁻¹. -1 The strong and broad absorption peak is due to the hydroxyl group O on the Dex molecule. The stretching vibration of H occurs at 2929 cm⁻¹. -1 The absorption peak at that point is on the C of the methylene group. The stretching vibration of H is 1429 cm.-1 With 1376cm -1 There are C O stretching vibration and The two absorption peaks are generated by the bending vibration coupling of OH; 1161 915cm -1 There are specific vibrational absorption peaks of the Dex sugar ring. These results indicate the presence of [specific vibrational absorption peaks] in Dex. OH、 CH2 and C O C.

[0081] The infrared spectrum of ODex, compared to Dex, is at 1728 cm⁻¹ -1 An absorption peak was added at this point, which is due to the aldehyde group. C=O The stretching vibration peak is at 2852 cm⁻¹. -1 An absorption peak was added at this point, which is the aldehyde group. C The presence of the stretching vibration peak of H, along with other characteristic peaks of Dex, indicates that the hydroxyl groups on the Dex molecule have been partially oxidized to aldehyde groups, and that the basic structures of ODex and Dex are still very similar.

[0082] Compared to ODex, the infrared spectrum of ODex / 5-ALA·HAL shows a lower spectral density (1728 cm⁻¹) because the aldehyde groups in ODex were not completely reacted. -1 2852 cm -1 The absorption peak at 1636 cm⁻¹ still exists, and a new peak at 1636 cm⁻¹ is added. -1 The absorption peak at that point is due to the stretching vibration of C=N on the imine bond, which indicates that 5-aminolevulinic hexyl ester was successfully grafted onto ODex via an imine bond.

[0083] (2) Characterization of proton nuclear magnetic resonance spectrum

[0084] The raw material Dex and the intermediate product ODex in Example 2 were sampled and analyzed by proton nuclear magnetic resonance spectroscopy. The obtained spectra are shown below. Figure 2 As shown. Dex and ODex 1 H In the NMR spectrum, at δ 3.4 The peaks between δ and 4.0 ppm represent H in the Dex molecule. 2 to H Comparing the proton NMR spectra of Dex before and after oxidation, it can be seen that ODex is significantly different from Dex at δ 5.5. A new peak belonging to the hemiacetal group appeared between δ5 and 8 ppm. This may be because after the hydroxyl group on Dex is partially oxidized to an acid group, the unoxidized hydroxyl group reacts with the adjacent acid group to form a cyclic hemiacetal structure, which is more stable than the free acid. At the same time, a new peak belonging to the aldehyde group appeared in ODex between δ9 and 9.5 ppm, indicating that the hydroxyl group on Dex is oxidized to an aldehyde group.

[0085] (3) Characterization by carbon nuclear magnetic resonance spectroscopy

[0086] The raw material Dex, the intermediate product ODex, and ODex / 5-ALA·HAL from Example 2 were sampled and analyzed by proton nuclear magnetic resonance spectroscopy. The obtained spectra are shown below. Figure 3 As shown. Comparison of Dex and ODex. 13 C NMR spectrum, located at δ175 A new peak at δ185 ppm was added, attributing to the aldehyde group, further confirming that the hydroxyl group on Dex was oxidized to an aldehyde group. Compared to ODex, ODex / 5-ALA·HAL shows that the aldehyde group in ODex did not react completely, hence the δ175 peak. The characteristic peaks of aldehyde groups are still present between δ185 ppm and δ150 ppm. A new characteristic peak attributable to imine bonds was added between δ155 ppm, and at δ160 ppm... A new characteristic peak attributable to the ester group was observed between δ170 ppm, which is a characteristic absorption peak of 5-ALA·HAL. This indicates that 5-ALA·HAL is grafted onto ODex via an imine bond.

[0087] (4) Ultraviolet spectrophotometric characterization

[0088] The raw material Dex, the intermediate product ODex, and ODex / 5-ALA·HAL from Example 2 were sampled and subjected to ultraviolet spectroscopy. The obtained spectra are shown below. Figure 4 As shown. By Figure 3 It can be seen that the ultraviolet spectra of Dex and ODex are significantly different. Dex has a different ultraviolet spectrum in the scanning wavelength range (200 nm). There is no absorption peak within 600 nm, while ODex has an absorption peak at 280 nm, proving that Dex was successfully oxidized to ODex. Comparing the spectra of ODex and ODex / 5-ALA·HAL, it can be seen that ODex / 5-ALA·HAL has a significant absorption peak at 220 nm, proving that 5-ALA·HAL was successfully grafted onto ODex.

[0089] (5) X-ray diffraction analysis

[0090] X-ray diffraction (XRD) was performed on samples of the raw material Dex, the intermediate product Dex-CHO, and ODex / 5-ALA·HAL, respectively. The resulting spectra are shown below. Figure 5 As shown in the diagram, a strong diffraction peak at 2θ = 17.7° is clearly visible in the Dex spectrum. After the oxidation reaction, the peak at 2θ = 17.7° is significantly weakened in the Dex-CHO spectrum. This may be because the oxidation of some hydroxyl groups on the Dex sugar ring to aldehyde groups leads to a decrease in the overall crystallinity of Dex, thus weakening the characteristic crystallization diffraction peak of Dex, indicating that Dex was successfully oxidized to ODex. Comparing the spectra of ODex and ODex / 5-ALA·HAL, it can be seen that the peak at 2θ = 17.7° shifts to 2θ = 18.9°, indicating that the grafting of 5-ALA·HAL has a slight effect on the crystallinity of Dex-CHO, but the effect is not significant.

[0091] (6) Characterization of hydrogels

[0092] The scanning electron microscope (SEM) image of the cross-section of the lyophilized sample of the final product ODex / 5-ALA·HAL / AMK hydrogel prepared in Example 2 is shown below. Figure 6 As shown, the magnification is 100x (left image) and 1000x (right image). The 100x magnified image shows that the hydrogel has a uniformly distributed pore structure with good continuity; after magnification to 1000x, the pore structure is smooth and complete, and has a certain thickness, proving that the hydrogel has an excellent three-dimensional spatial structure.

[0093] (7) pH-responsive drug release of hydrogels

[0094] The release behavior of 5-aminolevulinic acid hexyl ester and amikacin from the intelligent antibacterial hydrogel in Example 2 of the study was investigated in PBS buffer (0.1M) at pH 5.0 and pH 7.4. The results are as follows: Figure 7 As shown in the figure, the drug release rate of this hydrogel in an acidic environment is significantly higher than that in a PBS buffer solution at pH 7.4. Within 72 hours, in a PBS buffer solution at pH 7.4, the cumulative release rate of 5-aminolevulinic acid hexyl ester in the hydrogel sample of Example 2 was approximately 4.8%, and the cumulative release rate of amikacin was approximately 5.7%; while in a PBS buffer solution at pH 5.0, the cumulative release rate of 5-aminolevulinic acid hexyl ester was approximately 49.2%, and the cumulative release rate of amikacin was approximately 53.6%. Within 15 days, in a buffer solution at pH 5.0, the cumulative release rate of 5-aminolevulinic acid hexyl ester was approximately 62.3%, and the cumulative release rate of amikacin was approximately 71.9%. These results fully demonstrate that the hydrogel has significant pH sensitivity and sustained-release properties, enabling on-demand release in infectious environments.

[0095] Example 3

[0096] A method for preparing a photosensitizer-in-antibiotic smart antibacterial hydrogel is provided, comprising the following steps:

[0097] (1) Same as Example 1

[0098] (2) Same as Example 1

[0099] (3) Weigh a certain amount of amikacin sulfate (AMK) sample and dissolve it in deionized water to prepare 58.5 mg / ml. Dissolve ODEX / 5-ALA·HAL in deionized water to prepare 100 mg / ml for later use. Mix the two solutions at a volume ratio of 500 ml: 800 ml with ODEX / 5-ALA·HAL:AMK, pour the mixture into a small glass slide, stir thoroughly, and then place it in a constant temperature shaking oven at 37°C for 50 seconds to form a hydrogel. After freeze-drying, the photosensitizer combined with antibiotic intelligent antibacterial hydrogel can be obtained.

[0100] Example 4

[0101] A method for preparing a photosensitizer-in-antibiotic smart antibacterial hydrogel is provided, comprising the following steps:

[0102] (1) Same as Example 1

[0103] (2) Same as Example 1

[0104] (3) Weigh a certain amount of amikacin sulfate (AMK) sample and dissolve it in deionized water to prepare 58.5 mg / ml. Dissolve ODEX / 5-ALA·HAL in deionized water to prepare 100 mg / ml for later use. Mix the two solutions at a volume ratio of 500 ml: 1000 ml (ODEX / 5-ALA·HAL:AMK), pour the mixture into a small glass slide, stir thoroughly, and then place it in a constant temperature shaking oven at 37°C for 20 seconds to form a hydrogel. After freeze-drying, the photosensitizer combined with antibiotic intelligent antibacterial hydrogel can be obtained.

[0105] Study the above embodiment 1 The in vitro antibacterial effect of the prepared hydrogels on Staphylococcus aureus (Gram-positive bacteria) was investigated as follows: The experiment was conducted in a 96-well plate with 9 groups and 5 replicates per group. Group A: 60 μL of ODex solution (100 mg / ml) was added to each well; Group B: 60 μL of ODEX / 5-ALA·HAL solution was added to each well (the 5-ALA·HAL content was equal to the amount of 5-ALA·HAL released by 60 μL of Example 2 hydrogel at pH 5.0 for 24 h); Group C: 60 μL of 5-ALA·HAL solution was added to each well (the 5-ALA·HAL content was equal to the amount of 5-ALA·HAL released by 60 μL of Example 2 hydrogel at pH 5.0 for 24 h); Group D: 60 μL of AMK solution was added to each well (the AMK content was equal to the amount of TOB released by 60 μL of Example 2 hydrogel at pH 5.0 for 24 h); Group E: 60 μL of ODex / AMK solution was added to each well (the AMK content was equal to the amount of TOB released by 60 μL of Example 2 hydrogel at pH 5.0 for 24 h). Under 0.0 conditions, the amount of AMK released in 24 hours was equal (the hydrogels were the same); Group F received 60 μL of Example 1 hydrogel per well; Group G received 60 μL of Example 2 hydrogel per well; Group H received 60 μL of Example 3 hydrogel per well; and Group I received 60 μL of Example 4 hydrogel per well. After each group's materials were added, 100 μL of Staphylococcus aureus bacterial suspension (approximately 10 μL) was added to each well. 4 The viable bacterial density was determined by plate count method after incubation at 37℃ for 24 h (CFU / mL) in a shaker at 37℃ for 24 h without any antibacterial material. The result was recorded as 100% for control. The test results are shown below. Figure 8 .

[0106] Figure 8 The results showed that, compared with the control group, the antibacterial activity of ODex alone was the weakest, while the antibacterial effects of the Free 5-ALA·HAL solution group and the Free AMK solution group were slightly inferior to those of the ODEX / 5-ALA·HAL and ODex / AMK groups, respectively. Compared with the control group, the hydrogel of Example 2 showed a significant synergistic effect of 5-ALA·HAL and AMK, which significantly improved the antibacterial effect compared with single drugs, demonstrating excellent antibacterial performance. (Comparison with Example 1) 4. Theoretically, the higher the AMK concentration, the better the antibacterial effect of the hydrogel. However, as the AMK concentration increases, the density of the formed hydrogel framework increases, which is less conducive to the release of drug molecules. Therefore, the antibacterial ability of the hydrogel is weakened to some extent. Therefore, with Example 1... 4. As the concentration of AMK increased, the antibacterial effect showed a trend of first increasing and then decreasing.

[0107] Study the above embodiment 1 The in vitro antibacterial effect of the prepared hydrogels against *Pseudomonas aeruginosa* (a Gram-negative bacterium) was tested using the same procedure as the *Staphylococcus aureus* antibacterial test described above, except that *Staphylococcus aureus* was replaced with *Pseudomonas aeruginosa*. The test results are shown below. Figure 9 .

[0108] Figure 8-9 Antibacterial results showed that hexyl 5-aminolevulinate had a strong bactericidal effect against Staphylococcus aureus, but its antibacterial effect against Pseudomonas aeruginosa was slightly weaker due to its weak ability to penetrate biological barriers. Amikacin, on the other hand, had a strong inactivation ability against Pseudomonas aeruginosa and its antibacterial spectrum included most Gram-negative bacteria. From the perspective of the antibacterial effect of the hydrogel, the combined use of hexyl 5-aminolevulinate and amikacin could effectively kill most Gram-positive and Gram-negative bacteria, eliminating bacterial colonies caused by various types of wounds. Furthermore, the synergistic effect of 5-ALA·HAL and AMK significantly enhanced the antibacterial effect, effectively preventing or treating bacterial infections of wounds.

Claims

1. A photosensitizer combined with an antibiotic intelligent antibacterial hydrogel, characterized in that, OAHAL was prepared by bonding 5-aminolevulinic acid hexyl ester hydrochloride to oxidized dextran 40 as a hydrogel matrix via Schiff base reaction; then crosslinked with amikacin; wherein: The mass ratio of oxidized dextran 40 to hexyl 5-aminolevulinate hydrochloride is 2.5-5:1; the mass ratio of OAHAL to amikacin is 1.3-1.5:

1. Adjust the pH of the reaction system to 7-7.4 to carry out the Schiff base reaction.

2. The antibacterial hydrogel according to claim 1, characterized in that, Oxidized dextran 40 was prepared by oxidizing dextran 40 with sodium periodate, wherein the mass ratio of dextran 40 to sodium periodate was 1.5-1:

1.

3. A method for preparing a photosensitizer combined with an antibiotic intelligent antibacterial hydrogel according to any one of claims 1-2, characterized in that, The main steps include: 1) Oxidized dextran 40 and the photosensitizer precursor 5-aminolevulinic acid hexyl ester hydrochloride were mixed and subjected to a Schiff base reaction. After dialyzing and lyophilization, the product OAHAL was obtained. The mass ratio of oxidized dextran 40 to 5-aminolevulinic acid hexyl ester hydrochloride was 2.5-5:

1. The pH of the reaction system was adjusted to 7-7.4 for the Schiff base reaction. The reaction formula is as follows: 2) The product OAHAL obtained in step 1) is cross-linked with amikacin, and after dialysis and lyophilization, a photosensitizer combined with antibiotic intelligent antibacterial hydrogel is obtained; wherein the mass ratio of OAHAL to amikacin is 1.3~1.5:

1.

4. The preparation method according to claim 3, characterized in that, In step 1), the preparation of oxidized dextran 40 includes the following steps: Dextran 40 and sodium periodate were dissolved separately in deionized water. The sodium periodate solution was added dropwise to the dextran solution under stirring. The reaction was carried out in the dark with stirring for a period of time. After the reaction was terminated, stirring was continued in the dark. Finally, the product was dialyzed and lyophilized to obtain the oxidation product ODex. The reaction formula is as follows: 。 5. The preparation method according to claim 4, characterized in that, The reaction time is 12-24 hours, and after the reaction is terminated, stirring should be continued in the dark for 2-3 hours.

6. The preparation method according to claim 3, characterized in that, In step 1), the Schiff base reaction is carried out at a temperature of 40-60°C for 3-4 hours.

7. The preparation method according to claim 3, characterized in that, In step 2), the crosslinking reaction takes 20 seconds to 10 minutes and the temperature is 36 to 37.5 degrees Celsius.

8. The application of the intelligent antibacterial hydrogel according to claim 1 in the preparation of antibacterial drugs, characterized in that, The antibacterial drug is simultaneously loaded with the photosensitizer 5-aminolevulinic acid hexyl ester hydrochloride and the antibiotic amikacin.

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