Preparation method of selective permeation enzyme carrier of pH-responsive membrane and application thereof

Abstract

The application is suitable for the field of nanobiological medicine technology, and provides a preparation method of a pH response membrane selective permeation enzyme carrier, which comprises the following steps: step 1, preparation of a membrane selective permeation carrier: ternary complexes formed by CB[8], MV and AHQO are used as structural units to assemble into a membrane selective permeation carrier; and step 2, preparation of an enzyme cascade catalytic carrier: glucose oxidase (GOx), ferroferric oxide (Fe3O4) nanenzyme and arginine (L-Arg) are coated according to the above preparation steps of the enzyme cascade catalytic carrier. The prepared selective permeation enzyme carrier is applied to the treatment of diabetic chronic wounds. The method provides a new method for multiple enzyme synergistic treatment of diabetic chronic wounds based on the pH response semi-permeable membrane carrier, and effectively improves the wound healing rate of diabetic mice.

Description

Technical Field

[0001] This invention belongs to the field of nanobiomedicine technology, and particularly relates to a method for preparing a pH-responsive membrane selective permease carrier and its application. Background Technology

[0002] The skin is the largest organ in the human body and a natural defense barrier. External stimuli such as burns, surgical incisions, and other injuries can disrupt the skin's structural integrity, leaving the body vulnerable to microbial invasion and fluid loss. Wounds can be categorized into chronic and acute wounds based on healing time. Acute wounds heal relatively quickly, while chronic wounds, due to physiological disturbances in the wound tissue, require prolonged healing. It is reported that by approximately 2024, global spending on chronic wound care products will exceed $22 billion annually. About 20% of diabetic patients suffer from chronic wounds, severely impacting their health and exacerbating their financial burden. Therefore, addressing chronic wounds in diabetic patients is an urgent matter.

[0003] In recent years, multi-enzyme cascade catalytic reactions have attracted widespread attention in medicine and artificial cells due to their excellent biocompatibility. However, achieving high-efficiency catalytic reactions requires stable supports. Currently, mesoporous silica and metal-organic frameworks are used to realize enzyme cascade catalytic reactions. Selectively permeable membrane supports, due to their confined environment, not only act as a natural barrier, protecting enzyme molecules within the cavity from degradation by the external environment, but also can encapsulate different enzymes, bringing them closer together spatially, shortening substrate transport distances, realizing multi-enzyme cascade catalysis, and enhancing synergistic effects and conversion efficiency. However, further improving the targeted recognition ability of the support, enabling it to selectively accumulate on the target, and mimicking the semi-permeable barrier of biological cell membranes are of great significance for realizing the medical applications of these supports.

[0004] In recent years, gas therapy has shown great potential in promoting macrophage polarization due to its free permeability. NO, CO, H2S, and SO2 are important endogenous cellular signaling molecules with wide applications in regulating the cardiovascular, immune, central, and peripheral nervous systems. The most common NO donors include organic nitrates, nitrites, metal-NO complexes, diazoxides, and S-nitrosothiols; common H2S donors include sodium sulfide and sodium hydrosulfide. Their storage and transport in gas molecules have been extensively studied. However, these donors, especially organic ones, are highly toxic to biological cells. Furthermore, uncontrolled release rates leading to extremely high gas concentrations can also cause cytotoxicity, severely hindering their application in wound healing. Therefore, developing biocompatible, intelligently released gas therapies is of positive significance for wound repair. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing a selective permease carrier for a pH-responsive membrane and its application, aiming to solve the problems mentioned in the background art.

[0006] The present invention is implemented as follows: a method for preparing a selective permease carrier for a pH-responsive membrane includes the following steps:

[0007] Step 1: Preparation of membrane-selective permeability support:

[0008] Using the ternary complex formed by CB[8], MV and AHQO as structural units, a membrane selective permeability carrier was assembled.

[0009] Step 2, Preparation of enzyme cascade catalytic carrier: Coagulate glucose oxidase (GOx), iron(III) oxide (Fe3O4) nanozyme and arginine (L-Arg) according to the above steps for preparing enzyme cascade catalytic carrier.

[0010] In a further technical solution, step 1 includes the following specific steps:

[0011] Step 1.1: Weigh 1.329g of CB[8] and dissolve it in 1 L of Milli-Q to prepare a solution with a concentration of 10. -3 1. Dissolve 0.352 g MV in 1 L Milli-Q to prepare a solution of concentration 10. -3 1.326 g of AHQO was dissolved in 1 L of DMF to prepare a concentration of 10. -3 Mother liquor 3 of M;

[0012] Step 1.2: Take 100 μL of mother solution 1 and mother solution 2 and add them to 10 mL of Milli-Q. After sonicating for 30 min, add 100 μL of mother solution 3 and sonicate for 30 min to obtain the membrane-selective permeable carrier. Place it in a chromatography cabinet at 4℃ for later use.

[0013] In a further technical solution, step 2 includes the following specific steps:

[0014] Step 2.1: Add 10 mg GOx, 10 mg Fe3O4, 50 mg L-Arg, and 10 mg 4-mercaptophenylboronic acid (4-MPBA) to 10 mL of Milli-Q, which are defined as mother liquors 4, 5, 6, and 7 respectively.

[0015] Step 2.2: Take 100 μL of mother solution 1 and mother solution 2 and add them to 10 mL of Milli-Q. After sonicating for 30 min, add 50 μL of mother solution 4, 100 μL of mother solution 5 and 50 μL of mother solution 6. After sonicating for 10 min, add 100 μL of mother solution 3. After sonicating for 30 min, add 100 μL of 4-MPBA and stir overnight to obtain the enzyme cascade catalytic carrier. Store it in a chromatography cabinet at 4℃ for later use.

[0016] Another objective of this invention is to apply a pH-responsive membrane selective permeable enzyme carrier in the treatment of diabetic chronic wounds, thereby improving the wound healing rate of diabetic chronic wounds through the synergistic catalysis of reactive oxygen species and gas molecules generated by glucose oxidase (GOx), iron(trioxide) (Fe3O4) nanozyme and arginine (L-Arg) encapsulated in the selective permeable enzyme carrier.

[0017] This invention provides a method for preparing a pH-responsive membrane selectively permeable enzyme carrier and its application. Based on the pH-responsive semi-permeable membrane carrier, a novel method for the synergistic treatment of chronic diabetic wounds is invented. Through the synergistic catalysis of glucose oxidase (GOx), iron(III) oxide (Fe3O4) nanozyme and arginine (L-Arg) encapsulated in the carrier, the reactive oxygen species and gas molecules generated have antibacterial and anti-inflammatory effects, effectively improving the wound healing rate in diabetic mice. Attached Figure Description

[0018] Figure 1 A schematic diagram illustrating the self-assembly of a permeable carrier for membrane preparation;

[0019] Figure 2 This is a schematic diagram of the enzyme cascade catalysis principle;

[0020] Figure 3 This is a schematic diagram showing the size changes of the carrier at pH = 5.5 and 7.0;

[0021] Figure 4 The diagram shows how the "on / off" switch of the carrier is adjusted under different pH conditions to control the enzyme cascade reaction inside the cavity.

[0022] Figure 5 Investigation of the antibacterial properties of a GOx / Fe3O4-loaded and 4-MPBA-modified support: a) Plate coating experiment; b) Membrane permeability; c) Membrane potential; d) Changes in ATP content.

[0023] Figure 6 In the figures, 'a' represents the wound healing process; 'b' is a photograph of the wound healing process; 'c' is the wound area; 'd' is the blood glucose concentration in the mouse; 'e' is the change in the mouse's body weight; and 'f' is the change in bacterial colonies at the wound site.

[0024] Figure 7 H&E staining provides histopathological assessment of wound healing in W1–W6 at days 0 (a) and 14 (b) after treatment; c shows the epidermal and dermal structures of W2 and W6; d shows CD68 immunofluorescence staining in W1–W6; e shows the quantitative histogram of CD68 density; f shows the NO release curves of membrane-selective permeability carriers at pH 5.5 and 7; gj shows the relative levels of TNF-α and IL-10 in W6 serum and wound tissue. Scale bars: 100 μm (black), 50 μm (white).

[0025] Figure 8 In the figure, ad represents the immunofluorescence staining of VEGF, Ki-67, CD31, αSMA, and Sirius red, respectively; ei represents the quantitative histograms of VEGF density, Ki67 density, microvessel density, collagen volume fraction (CVF), and collagen I / III ratio (scale bar: 50 μm, where: * P<0.05, ** P<0.01, *** P<0.001 compared with W2). Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0027] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0028] like Figure 1 and 2 The diagram illustrates a method for preparing a selective permease carrier for a pH-responsive membrane, as provided in one embodiment of the present invention, comprising the following steps:

[0029] Step 1: Preparation of membrane-selective permeability support:

[0030] Using the ternary complex formed by CB[8], MV and AHQO as structural units, a membrane with selected permeability is assembled. Specifically:

[0031] Step 1.1: Weigh 1.329 g CB[8] and dissolve it in 1 L Milli-Q to prepare a concentration of 10. -3 1. Dissolve 0.352 g of stock solution M in 1 L of Milli-Q to prepare a solution of concentration 10. -3 1.326 g of AHQO was dissolved in 1 L of DMF to prepare a concentration of 10. -3 Mother liquor 3 of M;

[0032] Step 1.2: Take 100 μL of mother solution 1 and mother solution 2 and add them to 10 mL of Milli-Q. After sonicating for 30 min, add 100 μL of mother solution 3 and sonicate for 30 min to obtain the membrane-selective permeable carrier. Place it in a chromatography cabinet at 4℃ for later use.

[0033] Step 2, Preparation of enzyme cascade catalytic carrier: Following the steps described above for preparing the enzyme cascade catalytic carrier, glucose oxidase (GOx), iron(III) oxide (Fe3O4) nanozyme, and arginine (L-Arg) are coated onto the carrier. Specifically:

[0034] Step 2.1: Add 10 mg GOx, 10 mg Fe3O4, 50 mg L-Arg, and 10 mg 4-mercaptophenylboronic acid (4-MPBA) to 10 mL of Milli-Q, which are defined as mother liquors 4, 5, 6, and 7 respectively.

[0035] Step 2.2: Take 100 μL of mother solution 1 and mother solution 2 and add them to 10 mL of Milli-Q. After sonicating for 30 min, add 50 μL of mother solution 4, 100 μL of mother solution 5 and 50 μL of mother solution 6. After sonicating for 10 min, add 100 μL of mother solution 3. After sonicating for 30 min, add 100 μL of 4-MPBA and stir overnight to obtain the enzyme cascade catalytic carrier. Store it in a chromatography cabinet at 4℃ for later use.

[0036] In this embodiment of the invention, the permeability of the membrane selection carrier is characterized as follows:

[0037] Because the quinoline helical oligomer in the AHQO molecular structure is pH-responsive, this part undergoes an unwinding process under acidic conditions, causing the nano-units to rearrange and increasing the carrier size. Simultaneously, the carrier transforms from non-permeable to semi-permeable. To prepare the same carrier, 1 mL of the test carrier was added to 1 mL of buffer solution at pH = 7 and pH = 5.5, respectively. Figure 3 The size changes of the support were observed under a scanning electron microscope (SEM). SEM showed that before expansion, the diameter was approximately 164 nm and the film thickness was approximately 29 nm. After expansion under acidic conditions, the diameter was approximately 342 nm and the film thickness decreased from 29 nm to 6 nm.

[0038] The "on / off" state of the carrier at pH = 5.5 and 7.0 was determined using the TMB indirect method. In a 500 µL reaction system, 0.2 M acetate / phosphate buffer (pH = 5.5 / 7.0), 100 µL of the GOx-loaded carrier, and 10 µL of 8 mM TMB were added and stirred. Then, 10 µL of horseradish peroxidase (HRP) and 10 µL of 1 M glucose were added, and the mixture was stirred and reacted for 3 min before measuring the absorbance at 652 nm. Figure 4 As shown, the GOx-loaded support exhibits a membrane-dependent "on / off" switch. It is "on" at pH 5.5 and "off" at pH 7.0, at which point the absorbance remains unchanged. Furthermore, four switchable catalytic cycles were observed to control enzyme kinetics with pH changes, supporting a mechanism for regulating membrane permeability.

[0039] One embodiment of the present invention provides the application of a pH-responsive membrane selective permeable enzyme carrier in the treatment of diabetic chronic wounds. Through the synergistic catalysis of glucose oxidase (GOx), iron tetroxide (Fe3O4) nanozyme and arginine (L-Arg) encapsulated in the selective permeable enzyme carrier, the antibacterial and anti-inflammatory effects of reactive oxygen species and gas molecules are generated, thereby improving the wound healing rate of diabetic chronic wounds.

[0040] As a preferred embodiment of the present invention, the antibacterial properties of a selective permeable enzyme carrier loaded with glucose oxidase (GOx), iron(III) oxide (Fe3O4) nanozyme and arginine (L-Arg) were investigated:

[0041] The experiment was divided into five groups: control group PBS (Control group), Fe3O4-loaded vector (Group I), GOx-loaded vector (Group II), GOx / Fe3O4-loaded vector (Group III), and GOx / Fe3O4-loaded vector modified with 4-MPBA (Group IV).

[0042] Because the •OH produced by the enzyme cascade reaction has strong oxidizing properties, it can oxidize and destroy bacterial membrane structure and membrane potential, affecting bacterial biosynthesis, thereby achieving a highly efficient antibacterial effect. Figure 5 According to the plate coating experiment, its minimum inhibitory concentration is 5 μg / mL.

[0043] Membrane permeability: Propidium iodide (PI) was used as a fluorescent indicator to test changes in membrane permeability. Staphylococcus aureus (S. aureus) (1×10⁻⁶) was used. 8The bacterial pellet was mixed with different samples (CFU / mL, 1 mL), incubated at 37°C for 1 h, centrifuged at 8000 rpm for 1 min, washed three times with PBS, and then PI dye (5 μL / tube) was added. The pellet was then incubated at 37°C for 1 h, centrifuged at 8000 rpm for 3 min, washed three times with PBS, and then resuspended in PBS for fluorescence measurement (excitation light: 488 nm, emission light: 530 nm).

[0044] Membrane potential: E. coli and S. aureus (10 8 CFU / mL, 1 mL) were treated with the membrane potential dye DiSC3(5) (8 μM, 10 μL / tube) in a 37°C incubator for 1 h, centrifuged at 8000 rpm for 1 min, and washed 3 times with PBS. Subsequently, different samples were mixed with bacterial culture and incubated at 37°C for 1 h. Then, they were centrifuged at 8000 rpm for 3 min, washed 3 times with PBS, and resuspended in PBS for fluorescence measurement (excitation: 622 nm, emission: 670 nm).

[0045] ATP detection: using E. coli and S. aureus (10 8 (CFU / mL, 1 mL) Follow the instructions on the ATP content assay kit.

[0046] like Figure 5 .b. Compared with the control group, the membrane permeability of E. coli and S. aureus was increased. Membrane damage in S. aureus was more severe than in E. coli, indicating the selectivity of 4-MPBA for S. aureus. The membrane potential measurements for both bacteria were similar to the membrane permeability results. Figure 5 .c). Figure 5 Further analysis of bacterial ATP content in .d cells revealed that the antibacterial effect is achieved by interfering with bacterial metabolism, specifically by inhibiting membrane potential-dependent ATP synthesis, ultimately leading to bacterial death.

[0047] In an embodiment of the present invention, animal experiments were conducted simultaneously, specifically:

[0048] A diabetic mouse model was established, and all animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of Hainan University. Animals were housed in animal facilities according to the Hainan University Animal Care Guidelines. BALB / c mice aged 6–8 weeks (weighing 18–22 g) were acclimatized for 5–7 days before the experiment. After five intraperitoneal injections of 70 mg / kg streptozotocin (STZ), blood was collected from the tail tip three times to measure blood glucose levels. A blood glucose concentration greater than 11.1 mmol / L was required for successful establishment of a diabetic mouse model. Figure 6The mouse blood glucose level was greater than 11.1 mmol / L and remained stable, indicating that the diabetic mouse model was successfully established.

[0049] like Figure 6 The successfully modeled diabetic mice were randomly divided into five groups: control (without diabetes) (W1), control (with diabetes) (W2), H2O2 (W3), GOx / Fe3O4-loaded nanoreactor (W4), GOx / Fe3O4-modified 4-MPBA-loaded nanoreactor (W5), and GOx / Fe3O4 / Arg-loaded 4-MPBA-modified nanoreactor (W6). A 6 mm diameter wound was created on the back of each mouse using a puncture biopsy instrument, and each wound was infected with *S. aureus* (50 μL, 1×10⁻⁶). 7 CFU·mL -1 This period was recorded as day 0 of the experiment. Over the next three days, mice were treated with different drugs at the wound site. Wound photographs were taken using a digital camera on days 1, 3, 7, and 14 to calculate the wound healing rate. Mouse body weight was continuously monitored during wound healing. Additionally, sterile swabs were used to collect samples from the wound site on days 1, 3, 7, and 14 for microbiological assessment. The swabs were then incubated in LB (Luria-Bertani) liquid medium for 6 hours, and the absorbance of the bacterial suspension at 600 nm was measured using a visible spectrophotometer to calculate the bacterial viability density.

[0050] Figure 6 As shown in .b, compared with the control group, W6 had a wound repair rate of nearly 100% after 14 days. Figure 6 The mice's body weight remained relatively stable, indicating that the drug used had no significant toxic side effects on the mice. The bacterial density at the wound site in the mice was measured... Figure 6 As shown in .f, similar to the results of wound healing, the number of bacteria on the wound surface is significantly reduced as the wound heals.

[0051] Histological and immunohistochemical analysis:

[0052] Wound tissue was collected at 1 day and 14 days and dehydrated by soaking in 4% polyoxymethylene solution. Samples were embedded in paraffin, cut into 5 mm thick sections, and stained with hematoxylin and eosin (H&E) as well as Masson's trichrome. On day 14, the wound tissue was assessed for Sirius red, CD31, CD68, vascular endothelial growth factor (VEGF), and α-smooth muscle actin (α-SMA) using immunofluorescence staining.

[0053] Inflammatory factor concentrations in wound tissue: Treated mice were sacrificed at 3, 7, and 14 days. Wound tissue was homogenized in 10 volumes of tissue extraction buffer. After centrifugation at 400 rpm for 15 min, the concentrations of TNF-α and IL-10 in serum and supernatant were determined by enzyme-linked immunosorbent assay (ELISA).

[0054] like Figure 7 HE staining (.ab) showed typical epidermal hyperplasia. Numerous infiltrating inflammatory cells (green arrows) and extensive nuclear necrosis (blue arrows) were observed in the control group. These phenomena delayed the healing process and led to shortened collagen fibers (23.1 μm) and impaired skin remodeling (red double-headed arrows). In contrast, the synergistic therapy induced almost complete epidermal recovery in W6, with the epidermis of W6 being as thin as that of W1 and showing no significant hyperplasia. The epidermal structure was relatively intact, with less inflammatory cell infiltration and skin regeneration phenomena (e.g., hair follicles) (cyan arrows). Furthermore, the length of collagen increased to 54.2 μm (orange double-headed arrows), 2.3 times longer than that of W2.

[0055] Assessing the inflammatory response of macrophage recruitment using CD68 immunofluorescence staining, such as Figure 7 As shown in .d and .e, CD68-positive cells gradually decreased in W4, W5, and W6, demonstrating effective suppression of the inflammatory response. Subsequent investigations of in vitro NO release and in vivo inflammatory cytokine levels revealed a significant increase in NO levels at pH = 5.5, indicating that the low pH and high glucose microenvironment at diabetic wound sites favors NO production. Figure 7 The study measured two inflammatory factors in wound tissue and blood: tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10). TNF-α is a typical pro-inflammatory factor secreted by M1 macrophages, while IL-10 is an anti-inflammatory factor secreted by M2 macrophages. The polarization of M1 macrophages to M2 macrophages plays an important role in the wound repair process. The significant decrease in TNF-α levels in W6 and the enhanced expression of IL-10 in W6 serum and wound tissue confirm that NO can act as a gaseous mediator, accelerating wound healing by promoting the conversion of macrophages from the pro-inflammatory M1 phenotype to the repair M2 phenotype.

[0056] VEGF is an important pro-angiogenic factor that exhibits mitotic and anti-apoptotic effects on endothelial cells and promotes vascular permeability and cell migration during wound healing. Figure 8 As shown in .a, a significant increase in VEGF levels was observed in W6, approximately three times higher than in W2. Furthermore, Ki-67 was positively correlated with VEGF expression, as shown in .a. Figure 8 Quantitative analysis in sections b and f showed that the expression level of Ki-67 was approximately 67 times higher than that in W2, indicating that the W6 group induced accelerated wound healing by activating cell proliferation. Figure 8 .c and 8.g, with further assessment of vascular maturation using αSMA / CD31 double-positive cells to determine the direct effect of VEGF on wound angiogenesis, showed that W6 had the highest vascular density compared to other groups (W2: ~6.2-fold, W3: ~2.3-fold, W4: ~1.7-fold, W5: ~1.6-fold), indicating that it improves wound healing by driving angiogenesis during granulation tissue formation. Masson trichrome staining further examined morphological changes in wound tissue, such as... Figure 8 The healed area of ​​W6 showed almost complete epithelialization and high collagen deposition density. Sirius red staining allowed visualization of collagen fiber maturation, such as... Figure 8 In .d and 8.i, type I collagen (mature) was stained orange-red, and type III collagen (immature) was stained yellow-green. Compared with other groups, the repair tissue of W6 showed a significantly higher collagen I / III ratio, indicating the formation of mature collagen and the progression of wound repair to remodeling.

[0057] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of a selectively permeable enzyme carrier coated with a pH-responsive membrane containing glucose oxidase, iron oxide, and arginine in the preparation of a drug for treating chronic wounds of diabetes, characterized in that, The application utilizes a pH-responsive membrane containing glucose oxidase, iron oxide, and arginine as a selectively permeable enzyme carrier to selectively permeate the hydroxyl radicals and NO generated by the synergistic catalysis of these enzymes to improve the wound healing rate of chronic diabetic trauma. The method for preparing the selective permeable enzyme carrier of the pH-responsive membrane coated with glucose oxidase, ferric oxide, and arginine includes the following steps: First, 1.329 g of CB[8] was weighed and dissolved in 1 L of Milli-Q to prepare a solution with a concentration of 10. -3 1. Dissolve 0.352 g of MV in 1 L of Milli-Q to prepare a solution of concentration 10. -3 1.326 g of AHQO was dissolved in 1 L of DMF to prepare a solution with a concentration of 10. -3 Mother liquor 3 of M; Then, 10 mg of glucose oxidase, 10 mg of Fe3O4, 50 mg of arginine, and 10 mg of 4-mercaptophenylboronic acid were added to 10 mL of Milli-Q solution, which were then designated as mother solutions 4, 5, 6, and 7, respectively. Finally, 100 μL of mother solution 1 and mother solution 2 were added to 10 mL of Milli-Q, and after sonication for 30 min, 50 μL of mother solution 4, 100 μL of mother solution 5 and 50 μL of mother solution 6 were added. After sonication for 10 min, 100 μL of mother solution 3 was added. After sonication for 30 min, 100 μL of mother solution 7 was added and stirred overnight to finally obtain a selective permeable enzyme carrier for pH-responsive membranes coated with glucose oxidase, iron tetroxide and arginine. Wherein, CB[8] is cucurbit[8]urea, and the structural formula of MV is as follows: ； The structural formula of AHQO is as follows: 。

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