NO donor-loaded silica gel tube capable of controllably releasing NO and preparation method of silica gel tube
By loading NO donors in medical silicone tubes, the stable NO release is achieved by using hydrogen bonding, which solves the problem of rapid release of catheter antibacterial agents and achieves long-term antibacterial effects.
Patent Information
- Application Number
- CN202510453848.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-11
- Publication Date
- 2025-08-22
AI Technical Summary
During the long-term use of existing medical catheters, the rapid release of antibacterial agents cannot maintain effective concentrations for a long time, resulting in an increase in the risk of bacterial infection.
The NO donor containing hydroxyl groups is loaded into a medical silica tube by solvent swelling-impregnation method. The NO donor molecules are firmly loaded in the three-dimensional network structure of silica gel to achieve stable and continuous NO release.
It has achieved long-term antibacterial activity, high antibacterial rate, and can continuously inhibit bacteria, solving the problem of bacterial infections that are prone to long-term use of medical catheters.
Smart Images
Figure CN120514931A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of silica gel materials, and in particular to a silica gel tube loaded with an NO donor capable of controllably releasing NO and a preparation method thereof. Background Art
[0002] Inserting biomedical devices such as intravascular catheters and urinary catheters into the body is an integral part of modern healthcare, especially for hospitalized patients. However, the long-term or frequent use of these medical devices increases the risk of bacterial infection. Bacterial biofilms adhere to the catheter surface, easily leading to related infections in the body. In the case of long-term use of medical catheters (such as medical silicone tubes), the antibacterial effect of medical catheters in long-term use environments is particularly important. However, existing medical catheters are generally coated with an antibacterial agent (such as an antibacterial agent containing silver or silver ions) on their surface to form an antibacterial coating. The antibacterial agent is rapidly released in the early stage and cannot maintain an effective concentration over a long period of time, making it prone to bacterial infection during long-term use. Summary of the Invention
[0003] In response to the problems raised in the background technology, the purpose of the present invention is to propose a silicone tube loaded with NO donor and capable of controlling the release of NO and a preparation method thereof. The prepared silicone tube loaded with NO donor and capable of controlling the release of NO can achieve stable and continuous NO release, maintain the antibacterial activity of the material for a long time, achieve a continuous antibacterial effect, and has a high antibacterial rate, thereby solving the problem of bacterial infection that is prone to occur during long-term use of medical catheters.
[0004] The above technical objectives of the present invention are achieved through the following technical solutions:
[0005] A method for preparing a silicone tube loaded with an NO donor capable of controllably releasing NO comprises the following steps:
[0006] Prepare NO donor-tetrahydrofuran solution: mix NO donor and tetrahydrofuran evenly to obtain NO donor-tetrahydrofuran solution;
[0007] Silicone tube dipping: dipping the silicone tube into the NO donor-tetrahydrofuran solution, taking out the silicone tube after swelling, washing, and vacuum drying to obtain a silicone tube loaded with NO donor capable of controllably releasing NO;
[0008] The NO donor is an NO donor containing a hydroxyl group in its chemical structure.
[0009] Furthermore, the concentration of the NO donor-tetrahydrofuran solution is 100 mg / mL.
[0010] To further illustrate, during the immersion process of the silicone tube, the silicone tube is immersed in the NO donor-tetrahydrofuran solution and allowed to swell continuously for 24 to 36 hours.
[0011] It is further explained that during the immersion process of the silicone tube, the immersion is carried out under low temperature conditions and in the dark.
[0012] To further illustrate, the preparation steps of the NO donor include:
[0013] Acetic anhydride was added to a pyridine solution containing D-penicillamine. After the mixture was stirred and reacted, it was washed with an HCl solution and extracted with CHCl3. The mixture was dried over anhydrous Na2SO4 and then filtered and dried to obtain a yellow solid. The yellow solid was washed with petroleum ether to obtain a light yellow solid, which was then dried under vacuum overnight to obtain a thiolactone intermediate product A.
[0014] 4-Hydroxyphenylethylamine was added to a solution of the thiolactone intermediate A in CHCl3; after stirring at room temperature, the mixture was dried; the crude product was purified by column chromatography to obtain the free thiol intermediate B;
[0015] In an ice-water bath, hydrochloric acid and concentrated H2SO4 are added to a solution of the free thiol intermediate B in MeOH; when the temperature of the reaction mixture is below 5°C, NaNO2 is dissolved in water and added to the reaction mixture, and then the ice-water bath is removed; after stirring, the reaction mixture is extracted with CH2Cl2; the organic layer is dried over anhydrous Na2SO4, filtered, and dried by suction. The product is dried in vacuo overnight in the dark to obtain a NO donor containing a hydroxyl group.
[0016] To further illustrate, the washing step includes: washing the silica gel tube with tetrahydrofuran three times.
[0017] To further illustrate, the silicone tube is a medical silicone tube with an inner diameter of 2 mm and an outer diameter of 3.2 mm.
[0018] A silicone tube loaded with an NO donor capable of controllably releasing NO is prepared using the method for preparing a silicone tube loaded with an NO donor capable of controllably releasing NO.
[0019] Compared with the prior art, the embodiments of the present invention have the following beneficial effects:
[0020] A NO donor containing hydroxyl groups is loaded into a medical silicone tube by a solvent swelling-impregnation method to obtain a silicone tube loaded with NO donor and capable of controlling the release of NO, which is capable of achieving long-term NO release. By preparing a NO donor-tetrahydrofuran solution and immersing the silicone tube in a NOD solution, a light green drug-loaded silicone tube is obtained. The hydroxyl groups in the NO donor form hydrogen bond interactions with the silicon-oxygen bonds (Si-O-Si) in the silica gel matrix, so that the NO donor molecules (NOD molecules) are firmly loaded in the three-dimensional network structure of the silica gel, thereby reducing the leaching of the NO donor and extending the NO release time. The silicone tube loaded with NO donor and capable of controlling the release of NO can achieve stable and continuous NO release, maintain the antibacterial activity of the material for a long time, achieve a continuous antibacterial effect, and have a high antibacterial rate, solving the problem of bacterial infection easily occurring with long-term use of medical catheters. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 This is a graph showing the in vitro NO release of the silicone tube of Example 1 of the present invention.
[0022] Figure 2 Figure 3 is the antibacterial performance graph of the blank silicone tube (control group) and the silicone tube of Example 1 of the present invention against (a) Staphylococcus aureus and (b) Escherichia coli after 24 hours, (c) and (d) are SEM images of Staphylococcus aureus co-cultured with the control group and the NOD-loaded silicone tube, respectively, and (e) and (f) are SEM images of Escherichia coli co-cultured with the control group and the NOD-loaded silicone tube, respectively.
[0023] Figure 3 This is a diagram of the cumulative leaching of the donor of the silicone tube of Example 1 of the present invention. DETAILED DESCRIPTION
[0024] A method for preparing a silicone tube loaded with an NO donor capable of controllably releasing NO comprises the following steps:
[0025] Prepare NO donor-tetrahydrofuran solution: mix NO donor and tetrahydrofuran evenly to obtain NO donor-tetrahydrofuran solution;
[0026] Silicone tube dipping: dipping the silicone tube into the NO donor-tetrahydrofuran solution, taking out the silicone tube after swelling, washing, and vacuum drying to obtain a silicone tube loaded with NO donor capable of controllably releasing NO;
[0027] The NO donor is an NO donor containing a hydroxyl group in its chemical structure (abbreviated as NOD).
[0028] A NO donor containing hydroxyl groups is loaded into a medical silicone tube by a solvent swelling-impregnation method to obtain a silicone tube loaded with NO donor and capable of controlling the release of NO, which is capable of achieving long-term NO release. By preparing a NO donor-tetrahydrofuran solution and immersing the silicone tube in a NOD solution, a light green drug-loaded silicone tube is obtained. The hydroxyl groups in the NO donor form hydrogen bond interactions with the silicon-oxygen bonds (Si-O-Si) in the silica gel matrix, so that the NO donor molecules (NOD molecules) are firmly loaded in the three-dimensional network structure of the silica gel, thereby reducing the leaching of the NO donor and extending the NO release time. The silicone tube loaded with NO donor and capable of controlling the release of NO can achieve stable and continuous NO release, maintain the antibacterial activity of the material for a long time, achieve a continuous antibacterial effect, and have a high antibacterial rate, solving the problem of bacterial infection easily occurring with long-term use of medical catheters.
[0029] Furthermore, the concentration of the NO donor-tetrahydrofuran solution is 100 mg / mL.
[0030] Specifically, the NO donor-tetrahydrofuran solution was prepared as follows: 1.5 mL of tetrahydrofuran was added to a bottle, 150.0 mg of NO donor was weighed and added to the bottle, and the mixture was mixed to obtain a stable and uniform 100 mg / mL NO donor-tetrahydrofuran solution.
[0031] To further illustrate, during the immersion process of the silicone tube, the silicone tube is immersed in the NO donor-tetrahydrofuran solution and allowed to swell continuously for 24 to 36 hours.
[0032] When the silicone tube is immersed in the NO donor-tetrahydrofuran solution, the silicone tube is allowed to swell continuously for 24 to 36 hours, thereby ensuring that the silicone tube fully adsorbs the NO donor molecules.
[0033] It is further explained that during the immersion process of the silicone tube, the immersion is carried out under low temperature conditions and in the dark.
[0034] The NO donor is sensitive to light and heat, so the impregnation process is carried out at low temperature and in the dark. Specifically, the low temperature condition is placed in a refrigerator at 4°C.
[0035] To further illustrate, the preparation steps of the NO donor include:
[0036] Acetic anhydride (8.82 mL, 93.96 mmol) was added to a pyridine (15 mL) solution containing D-penicillamine (5.001 g, 33.56 mmol), and the mixture was stirred at room temperature for 20 hours. After the reaction, the mixture was washed with HCl solution (1.0 M, 50 mL x 3), extracted with CHCl (100 mL), dried over anhydrous NaSO, and filtered to dryness to obtain a yellow solid. The yellow solid was washed with petroleum ether to obtain a light yellow solid, which was then dried under vacuum overnight to obtain the thiolactone intermediate A. The synthetic route is as follows:
[0037]
[0038] To a solution of thiolactone intermediate A (700.0 mg, 4.046 mmol) in CHCl₃ (30 mL) was added 4-hydroxyphenylethylamine (4.855 mmol, 1.2 eq.); after stirring at room temperature for 24 hours, the mixture was dried; the crude product was purified by column chromatography (1:35 MeOH-CH₂Cl₂) to afford the free thiol intermediate B (white solid). The synthetic route is as follows:
[0039]
[0040] To a solution of the free thiol intermediate B (200 mg, 0.6452 mmol) in MeOH (10 mL) in an ice-water bath, hydrochloric acid (10 mL, 1.0 M) and concentrated H2SO4 (2 mL) were added. When the reaction mixture was cooled below 5°C, NaNO2 (3 eq.) was dissolved in water (2 mL) and added to the reaction mixture, and the ice-water bath was removed. After stirring for 1 hour, the reaction mixture was extracted with CH2Cl2 (5 mL x 3). The organic layer was dried over anhydrous Na2SO4, filtered, and dried overnight in a vacuum oven in the dark to yield the hydroxyl-containing NO donor (NOD). The synthetic route is as follows:
[0041]
[0042] The chemical structural formula of the hydroxyl-containing NO donor (NOD) is:
[0043]
[0044] To further illustrate, the washing step includes: washing the silica gel tube with tetrahydrofuran three times to remove the free donor molecules physically adsorbed on the surface of the silica gel tube.
[0045] To further illustrate, the silicone tube is a medical silicone tube with an inner diameter of 2 mm and an outer diameter of 3.2 mm.
[0046] A silicone tube loaded with an NO donor capable of controllably releasing NO is prepared using the method for preparing a silicone tube loaded with an NO donor capable of controllably releasing NO.
[0047] For ease of understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.
[0048] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. All reagents or instruments used without specifying the manufacturer are commercially available conventional products.
[0049] Example 1
[0050] A method for preparing a silicone tube loaded with an NO donor capable of controllably releasing NO comprises the following steps:
[0051] Prepare NO donor-tetrahydrofuran solution: add 1.5 mL of tetrahydrofuran (AR Aladdin) to a bottle, weigh 150.0 mg of NO donor and add it to the bottle, mix well to obtain a stable and uniform 100 mg / mL NO donor-tetrahydrofuran solution;
[0052] Silicone tube immersion: A medical-grade silicone tube (inner diameter × outer diameter: 2.0 mm × 3.2 mm) was immersed in the NO donor-tetrahydrofuran solution. After continuous swelling for 24 h in a refrigerator at 4°C in the dark, the silicone tube was removed and washed three times with tetrahydrofuran (AR Aladdin) to remove free NO donor molecules physically adsorbed on the surface. The tube was then dried in a vacuum drying oven for 4 h to completely remove the residual solvent, resulting in a light green silicone tube loaded with NO donor for controlled NO release (SR-NOD).
[0053] The preparation steps of the NO donor are as follows:
[0054] Acetic anhydride (8.82 mL, 93.96 mmol) was added to a pyridine (15 mL) solution containing D-penicillamine (5.001 g, 33.56 mmol), and the mixture was stirred at room temperature for 20 hours. After the reaction, the mixture was washed with HCl solution (1.0 M, 50 mL x 3), extracted with CHCl (100 mL), dried over anhydrous NaSO, and filtered to dryness to obtain a yellow solid. The yellow solid was washed with petroleum ether to obtain a light yellow solid, which was then dried under vacuum overnight to obtain the thiolactone intermediate A. The synthetic route is as follows:
[0055]
[0056] To a solution of thiolactone intermediate A (700.0 mg, 4.046 mmol) in CHCl₃ (30 mL) was added 4-hydroxyphenylethylamine (4.855 mmol, 1.2 eq.); after stirring at room temperature for 24 hours, the mixture was dried; the crude product was purified by column chromatography (1:35 MeOH-CH₂Cl₂) to afford the free thiol intermediate B (white solid). The synthetic route is as follows:
[0057]
[0058] To a solution of the free thiol intermediate B (200 mg, 0.6452 mmol) in MeOH (10 mL) in an ice-water bath, hydrochloric acid (10 mL, 1.0 M) and concentrated H2SO4 (2 mL) were added. When the reaction mixture was cooled below 5°C, NaNO2 (3 eq.) was dissolved in water (2 mL) and added to the reaction mixture, and the ice-water bath was removed. After stirring for 1 hour, the reaction mixture was extracted with CH2Cl2 (5 mL x 3). The organic layer was dried over anhydrous Na2SO4, filtered, and dried overnight in a vacuum oven in the dark to yield the hydroxyl-containing NO donor (NOD). The synthetic route is as follows:
[0059]
[0060] The silicone tube prepared in Example 1 was subjected to NO release test, antibacterial test and donor leaching test:
[0061] 1. NO release test.
[0062] The NO release characteristics of silicone tubing loaded with an NO donor for controlled NO release were measured using a NO analyzer (Model 42i, Thermo Scientific, USA). Before the test, a 1 cm long piece of silicone tubing (inner diameter × outer diameter: 2.0 mm × 3.2 mm) was cut and weighed, then placed in a brown quartz reactor. 2 mL of PBS buffer was drawn up using a syringe and slowly added to the reactor. Finally, the reactor was placed in a 37°C water bath. Throughout the test, to prevent external interference with NO release and ensure the stability of the test environment, a nitrogen purge mass flow rate of 0.01 kg / h was maintained.
[0063] In order to explore the characteristics of NO release in vitro from the silicone tube loaded with NOD, chemiluminescence was used to evaluate the release. The silicone tube was placed in 2 mL of PBS solution at a constant temperature of 37°C to simulate the physiological fluid environment in the body. The amount of NO released was monitored in real time by chemiluminescence. The experimental results are as follows: Figure 1As shown in Figure 2, the NOD-loaded silicone tube exhibited excellent NO release performance, with NO release lasting up to 299 hours and a total NO release rate of 22.07 ppb / mg. A detailed analysis of the release curve revealed a rapid increase in NO release during the initial phase (0-80 hours), followed by a gradual decrease, with the release rate dropping sharply from 0.5 ppb / h to 0.025 ppb / h. This phenomenon occurs because some NOD molecules have not yet formed interactions with the silica gel. Upon contact with the PBS solution, the silica gel absorbs water and swells, causing some NOD to dissolve and release NO. A stable sustained-release plateau (0.025 ppb / h) was maintained during the sustained-release phase (80-299 hours). This is attributed to the hydrogen bonding network formed between the hydroxyl groups in the NOD molecules and the siloxane (Si-O-Si) bonds in the silica gel matrix. This intermolecular interaction securely anchors the NOD molecules within the three-dimensional silica gel network, effectively reducing the drug-medium contact area and prolonging the release period. As a powerful biomediator, NO has numerous important physiological activities in vivo. It plays a key role in preventing thrombosis. It can promote vasodilation, keep blood vessels in good elasticity and patency; effectively inhibit platelet adhesion and aggregation, prevent abnormal accumulation of platelets in the blood vessel wall to form thrombus; at the same time, it can also effectively prevent and control the proliferation of smooth muscle cells, maintain the normal structure and function of the blood vessel wall. By loading the donor molecule NOD into silica gel, it can not only achieve long-term stable release of NO donor to achieve long-term antibacterial effect, but also has positive significance for the treatment of thrombosis.
[0064] The NO donor in this application is a typical S-nitrosothiol (RSNO) compound (hydrogen bond NO donor). RSNO can release NO by photolysis and pyrolysis, and simultaneously produce corresponding disulfide bond substances (RSSR). In order to eliminate the interference of photolysis factors on the experimental results, the NO release experiment was carried out in a constant temperature water bath environment of 37°C under a light-proof environment. When nitrosothiol is exposed to 37°C ambient heat, the S-NO bond inside its molecule will break to form NO and the corresponding disulfide bond. After studying and analyzing the in vitro release characteristics of the silicone tube loaded with NO donors that can controllably release NO, the silicone tube loaded with NO donors that can controllably release NO of the present invention can release NO, which is of great help in antibacterial and anti-thrombotic effects and improving the biocompatibility of medical devices.
[0065] 2. Antibacterial test.
[0066] Antimicrobial testing: First, strains stored at -80°C were thawed and inoculated onto nutrient agar plates and incubated in a 37°C incubator for 24 hours. One or two colonies were then picked with a loop and placed into 100 mL of sterile MRS broth, shaken, and incubated in a 37°C shaker for 24 hours. A 2 cm long blank silicone tube and an SR-NOD (2.0 mm × 3.2 mm ID) were placed in a centrifuge tube, 1 mL of the bacterial solution was added, and the mixture was incubated in a 37°C incubator for 24 hours. The sample-bacteria mixture was then removed from the incubator, and the silicone tube, along with the bacterial solution, was transferred to a centrifuge tube containing fresh PBS buffer. A ten-fold serial dilution method was used, and 50.0 μL of the dilution was plated onto nutrient agar for the dilution-smear test. Each sample group was tested in triplicate. After incubation at 37°C for 24 hours, the number of colonies on each plate was accurately recorded to verify antimicrobial activity.
[0067] Biofilm experiment: Escherichia coli and Staphylococcus aureus were co-cultured with blank silicone tubes and SR-NOD in a 37°C constant temperature incubator in the dark for 24 hours. The co-cultured bacterial liquid was collected and centrifuged at 5000 rpm for 20 minutes. The supernatant was discarded, and the bottom sediment was taken and washed twice with PBS buffer (20 minutes each centrifugation). Subsequently, 2.5% glutaraldehyde was added to fix the bacteria at 4°C overnight. The glutaraldehyde was removed by centrifugation the next day, and the samples were dehydrated with gradient ethanol / water mixtures (ethanol volume fractions of 30%, 50%, 70%, 90% and 100% respectively) for 15 minutes, and centrifuged for 20 minutes each time. Finally, the samples were treated with gold spraying at 30 mA for 1 minute, and the morphological changes of Escherichia coli and Staphylococcus aureus were observed by scanning electron microscopy (SEM).
[0068] The present invention selected a blank silicone tube and the silicone tube prepared in Example 1, and conducted antibacterial tests on two typical bacteria, Staphylococcus aureus and Escherichia coli. The blank silicone tube and the silicone tube loaded with NOD used have uniform specifications, with a length of 2 cm, an inner diameter of 2 mm, and an outer diameter of 3.2 mm. At the beginning of the test, the above two silicone tubes were respectively placed in the bacterial solution containing Staphylococcus aureus and Escherichia coli, and co-cultured for 24 hours at a constant temperature of 3 ° C. After the culture was completed, the silicone tube was carefully taken out, and the antibacterial activity of the silicone tube was measured by the dilution coating method.
[0069] The antibacterial test results of silicone tube are presented in Figure 2(a) and (b) of the figure. It can be seen intuitively from the data in the figure that after the addition of the donor molecule NOD, the NO-releasing silicone tube showed a significant inhibitory effect on both Staphylococcus aureus and Escherichia coli. Specifically, compared with the control group, the number of Staphylococcus aureus under the action of the SR-NOD silicone tube was significantly reduced by 2.3 orders of magnitude, and its antibacterial rate was calculated to be as high as 99.50%. For Escherichia coli, the SR-NOD silicone tube also performed well, with the number of bacteria reduced by 2.6 orders of magnitude and the antibacterial efficiency reaching 99.77%. The strong antibacterial ability of NO does not come from its own molecular structure, but is attributed to its high instability within the cell. In the complex environment within the cell, NO easily reacts chemically with oxygen or reactive oxygen intermediates (such as peroxynitrite), thereby generating a series of substances with high oxidative activity. These newly generated oxidative active substances will further trigger the oxidation reaction of membrane proteins, directly causing serious damage to the cell membrane. Crucially, once NO successfully penetrates the cell membrane, the resulting series of reaction products leads to DNA oxidation, ultimately causing irreversible damage to the DNA strands, severely disrupting the cell's normal physiological functions and the stability of its genetic information. Furthermore, over a 24-hour period, SR-NOD maintained NO release levels above 0.2 ppb / mg. The remarkable antibacterial efficacy of SR-NOD is primarily due to the high amount of NO released by the donor molecule, NOD, during the initial phase.
[0070] In order to explore the intrinsic antibacterial mechanism of SR-NOD in a more in-depth and comprehensive manner, we further used biofilm images to verify its antibacterial effect. Figure 2 From the observations of (c) and (e), it can be found that the Staphylococcus aureus cultured with the blank silicone tube showed a complete cell morphology, with clear edge contours, a smooth surface, and maintained a good physiological state. Similarly, the Escherichia coli cultured with the blank silicone tube also showed a typical rod-shaped appearance, with a complete morphology and no obvious damage. However, when observing Figure 2 When (d) and (f) were taken, completely different pictures were presented. Staphylococcus aureus and Escherichia coli co-cultured with SR-NOD showed obvious wrinkles on their surfaces, and some bacteria even had ruptured cell membranes, causing leakage of substances inside the cells. Combining the antibacterial activity test results of bacterial surfaces and scanning electron microscope images, it was concluded that SR-NOD can be used as a highly effective and long-lasting antibacterial agent. With its excellent antibacterial properties and stable effects, it excels in combating various common pathogenic bacteria. In the field of antibacterial medical devices, whether it is used to manufacture surgical instruments, implants, or daily medical protective supplies, it has broad application prospects and is expected to bring new breakthroughs in antibacterial protection in the medical field.
[0071] 3. Donor leaching test.
[0072] Cut each silicone tubing sample (blank silicone tubing and SR-NOD) into 1 cm lengths, weigh them, and place them in a brown vial containing a mixture of acetonitrile and water (2 mL, 1:9 volume ratio). Three replicates were run for each sample. Each vial was placed at 3°C in a dark environment. The acetonitrile and water mixture was removed every 12 or 24 hours and its absorbance at 339 nm was measured using a UV-visible spectrophotometer. The concentration of NOD leached at this time was calculated based on a standard curve. Simultaneously, a fresh solvent mixture was replaced and placed again at 3°C in a dark environment. This process was repeated until no more donor leaching occurred.
[0073] Currently, NO-releasing materials have attracted much attention due to their great potential in antibacterial and tissue repair. However, the problem of NO donor leaching has become one of the key challenges that need to be solved in the practical application of such materials. The uncontrolled leaching of NO donors from the material is likely to cause a series of negative effects, seriously restricting the performance and application prospects of NO-releasing materials. In clinical medical applications, once the NO donor leaches uncontrollably, the NO concentration in the local area will rise rapidly, which can easily exceed the safe range that cells can tolerate, thereby causing significant cytotoxicity. This is because excessive NO will react with the O 2- A chemical reaction occurs, generating highly oxidizing peroxynitrite. This substance can cause irreversible oxidative damage to important biomolecules such as cellular lipids, proteins, and DNA. Simultaneously, premature depletion of the NO donor prevents the material from achieving sustained and stable NO release, which undoubtedly severely impacts its therapeutic efficacy and makes it difficult to meet the long-term therapeutic needs of clinical applications.
[0074] Given the importance and urgency of NO donor leaching, the chemical leaching behavior of NOD in silicone tubing was tested. The absorbance of the leached solution was measured using a UV-visible spectrophotometer, and the NOD concentration in the hydrogel sample was calculated using a NOD standard curve. Figure 3 The experimental results presented intuitively reflect the leaching of SR-NOD in the silicone tube. On the first day of the experiment, the leaching amount of SR-NOD reached 0.00693mmol / g. As the immersion time increased, by the 7th day, the total amount of chemical leaching accumulated to 0.00963mmol / g polymer, and there was almost no NOD leaching after that. It was calculated that the leaching amount on the first day accounted for 80.0% of the total leaching amount. This shows that in the early stage of the experiment, the chemical leaching rate was relatively high, but in the following few days, the leaching rate of the donor showed a significant slowdown trend, only 0.00045mmolg- 1 d -1 .
[0075] This trend in leaching rate is of significant significance, particularly when analyzed in conjunction with the antibacterial properties of NO. In the early stages of testing, the leaching of an appropriate amount of SR-NOD can trigger a rapid and sustained release of NO. Leveraging NO's potent antibacterial properties, NO can rapidly inhibit bacterial growth and reproduction. For example, NO can effectively inhibit bacterial growth and reproduction by disrupting the bacterial respiratory chain and cell membrane integrity, effectively controlling bacterial numbers in the early stages and preventing rapid proliferation and spread, thus creating favorable conditions for subsequent antibacterial processes. Over time, hydrogen bonding between the -OH groups on the benzene ring of the donor molecule NOD and the O-Si-O groups of the silicone tubing plays a key role in inhibiting donor molecule leakage, resulting in a more sustained and stable NO release process. This stable and sustained NO release pattern maintains the material's antibacterial activity over a long period of time, continuously inhibiting bacterial regeneration and resuscitation. This provides a strong guarantee for the stability and sustainability of SR-NOD in antibacterial applications, giving it broad application prospects in the field.
[0076] This study successfully loaded a hydroxyl group of NOD into a silicone tubing via a solvent swelling-impregnation method. Hydrogen bonding interactions between the NOD and the silicone structure enabled controlled NO release. The resulting silicone tubing offers dual advantages: significantly reducing the risk of NO donor leakage and significantly extending the duration of NO release from the SR-NOD. Chemiluminescence analysis of the NO release behavior of the SR-NOD demonstrated sustained and stable NO release for up to 299 hours under simulated physiological conditions. Furthermore, NO donor leakage testing further validated the key role of hydrogen bonding in mitigating chemical leaching. SR-NOD exhibited exceptional antibacterial properties, exceeding 99% against both Escherichia coli and Staphylococcus aureus, and exhibited significant anti-biofilm activity. In summary, silicone tubing loaded with NO donors for controlled NO release can address the problem of bacterial infections associated with long-term or frequent catheter use in hospitals and holds broad application prospects in the biomedical field.
[0077] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.
Claims
1. A method for preparing a silicone tube loaded with NO donor capable of controlling the release of NO, characterized in that: The following steps are involved: Prepare NO donor-tetrahydrofuran solution: mix NO donor and tetrahydrofuran evenly to obtain NO donor-tetrahydrofuran solution; Silicone tube dipping: dipping the silicone tube into the NO donor-tetrahydrofuran solution, taking out the silicone tube after swelling, washing, and vacuum drying to obtain a silicone tube loaded with NO donor capable of controllably releasing NO; The NO donor is an NO donor containing a hydroxyl group in its chemical structure.
2. The method for preparing a silicone tube loaded with NO donor capable of controlling the release of NO according to claim 1, characterized in that: The concentration of the NO donor-tetrahydrofuran solution is 100 mg / mL.
3. The method for preparing the silicone tube loaded with NO donor capable of controlling the release of NO according to claim 1, characterized in that: During the silicone tube immersion process, the silicone tube is immersed in the NO donor-tetrahydrofuran solution and continues to swell for 24 to 36 hours.
4. The method for preparing a silicone tube loaded with NO donor capable of controlling the release of NO according to claim 1, characterized in that: During the dipping process of the silicone tube, the dipping is carried out at a low temperature and in the dark.
5. The method for preparing a silicone tube loaded with NO donor capable of controlling the release of NO according to claim 1, characterized in that: The preparation steps of the NO donor include: Acetic anhydride was added to a pyridine solution containing D-penicillamine. After the mixture was stirred and reacted, it was washed with an HCl solution and extracted with CHCl3. The mixture was dried over anhydrous Na2SO4 and then filtered and dried to obtain a yellow solid. The yellow solid was washed with petroleum ether to obtain a light yellow solid, which was then dried under vacuum overnight to obtain a thiolactone intermediate product A. 4-Hydroxyphenylethylamine was added to a solution of the thiolactone intermediate A in CHCl3; after stirring at room temperature, the mixture was dried; the crude product was purified by column chromatography to obtain the free thiol intermediate B; In an ice-water bath, hydrochloric acid and concentrated H2SO4 are added to a solution of the free thiol intermediate B in MeOH; when the temperature of the reaction mixture is below 5°C, NaNO2 is dissolved in water and added to the reaction mixture, and then the ice-water bath is removed; after stirring, the reaction mixture is extracted with CH2Cl2; the organic layer is dried over anhydrous Na2SO4, filtered, and dried by suction. The product is dried in vacuo overnight in the dark to obtain a NO donor containing a hydroxyl group.
6. The method for preparing a silicone tube loaded with NO donor capable of controlling the release of NO according to claim 1, characterized in that: The washing step comprises: washing the silica gel tube with tetrahydrofuran three times.
7. The method for preparing a silicone tube loaded with NO donor capable of controlling the release of NO according to claim 1, characterized in that: The silicone tube is a medical silicone tube with an inner diameter of 2 mm and an outer diameter of 3.2 mm.
8. A silicone tube loaded with NO donor capable of controlling the release of NO, characterized in that: The silicone tube is prepared using the method for preparing a silicone tube loaded with an NO donor capable of controlling the release of NO as described in any one of claims 1 to 7.