A method for preparing cationic polyrotaxane and its application

Abstract

This invention discloses a cationic ring-locked polyrotaxane, wherein the polyrotaxane comprises an axial molecule, a ring molecule, a capped structure, and a linkage structure formed by the chemical reaction of coaxially adjacent ring molecules with hydroxyl groups on adjacent ring molecules; the axial molecule is polyethylene glycol or a polyethylene glycol block copolymer; the ring molecule is cyclodextrin; and the ring molecule is modified with cationic ligands. The results of this invention show that when the polyrotaxane ring molecule is partially ring-locked, a balance between the dynamics and synergistic effects of the cationic ligands on the ring molecule can be achieved, significantly improving the bactericidal ability of the cationic ring-locked polyrotaxane. This allows the cationic antibacterial polymer to possess high antibacterial efficacy under low cation density conditions without causing toxicity to normal cells. This invention aims to develop a cationic ring-locked polyrotaxane based on a polyrotaxane structure, which, through innovative molecular design, improves the bactericidal effect and has significant clinical application potential.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials, specifically to a method for preparing cationic polyrotaxane and its application. Background Technology

[0002] In the interdisciplinary field of modern materials science and biomedicine, how to effectively and safely combat bacterial infections has always been a crucial and highly regarded topic. The bacterial cell membrane is a key target in the treatment of drug-resistant bacteria; its relatively stable structure and resistance to mutation have made it a significant area of ​​research in antibacterial strategies in recent years. In particular, the electronegative nature of the bacterial cell membrane provides a unique entry point for antibacterial therapy; the negative charge on the bacterial cell membrane creates a strong attraction for positively charged molecules. Therefore, cationic antimicrobial polymers have become one of the most promising alternatives to antibiotics. These cationic polymers can induce membrane rupture or morphological changes through electrostatic interactions with the cell membrane, disrupting its integrity and thus inhibiting or killing bacteria. To enhance the antibacterial properties of cationic antimicrobial polymers, a high cation density is usually required. However, high cation density often comes with high cytotoxicity, even leading to an overreaction of the immune system and causing severe toxic side effects. Therefore, how to enhance antibacterial efficacy while reducing toxicity to host cells is a key issue in the research of cationic antimicrobial polymers.

[0003] Polyrotaxane, as a typical representative of artificially synthesized molecular machines, offers a new approach to solving this problem due to its unique molecular structure. Polyrotaxane consists of an axial molecule and multiple macrocyclic molecules. The axial molecule passes through the cavity of the macrocyclic molecules, and the ring molecules are connected to the axial molecule through mechanical interlocking. The two ends of the macrocyclic molecules are sealed by large-volume end-capping agents to prevent the ring molecules from slipping out, thus forming a unique mechanical structure. The dynamic structure of polyrotaxane gives it significant advantages in antibacterial therapy. Unlike traditional static cationic polymers, this dynamically changing molecular structure allows polyrotaxane to interact more flexibly with bacterial cell membranes. However, currently, further improving the antibacterial properties of polyrotaxane can only be achieved by excessively increasing the number of cationic ligands to increase the cationic ligand density, thereby enhancing its antibacterial performance. Patent CN114949253A discloses a dual-drug-loaded polyrotaxane nanoparticle drug delivery system, its preparation method, and its application. It discloses a method using mesoporous polydopamine (mPDA) as the core, modifying the surface of cationic polyrotaxane (PR-PHEA) with molecular mobility to obtain cationic polyrotaxane nanomaterials (mPDA@PR-PHEA), resulting in a polyrotaxane nanoparticle drug delivery system co-loaded with clotrimazole and NO. This dual-drug-loaded polyrotaxane nanoparticle drug delivery system can effectively accelerate and enhance the contact between the NO drug delivery system and fungi, strengthen the interaction between the material itself and fungi, and improve the bioavailability of NO and the loaded drug, further realizing the synergistic effect of NO and the drug, and showing therapeutic effects on Candida albicans and related diseases. However, this patent only discloses how to utilize a polyrotaxane nanoparticle drug delivery system to improve the bioavailability and synergistic effects of NO and the loaded drug to achieve better antibacterial efficacy, rather than focusing on regulating the cyclic molecular mobility of polyrotaxane itself to achieve a dynamic and synergistic balance with its modified cationic ligands, thereby enabling the cationic polyrotaxane itself to possess superior antibacterial properties and less cytotoxicity. Therefore, providing a highly efficient and low-toxicity cationic antibacterial polymer is of great significance in promoting the development of antibacterial strategies towards greater efficiency and safety. Summary of the Invention

[0004] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide a cationic ring-locked polyrotaxane.

[0005] A second object of the present invention is to provide a method for preparing cationic ring-locked polyrotaxane as described above.

[0006] A third object of the present invention is to provide the use of the above-mentioned cationic ring-locked polyrotaxane in antibacterial applications or in the preparation of antibacterial products.

[0007] The above-mentioned objective of this invention is achieved through the following technical solution:

[0008] This invention provides a cationic ring-locked polyrotaxane, the polyrotaxane comprising an axial molecule, a ring molecule, a capped structure, and a linkage structure formed between coaxially adjacent ring molecules through a chemical reaction with hydroxyl groups on adjacent ring molecules; the axial molecule is polyethylene glycol or a polyethylene glycol block copolymer; the ring molecule is cyclodextrin; and the ring molecule is modified with a cationic ligand.

[0009] This invention synthesizes polyrotaxane using axial molecules, ring molecules, and end-capping agents. A bridging agent controls the dynamics of the ring molecules, and cationic ligands are then modified onto the ring-locked polyrotaxane. By utilizing the dynamics of the polyrotaxane ring molecules, the carrier structure of the cationic ligands is altered, enhancing their dynamics and synergistic effects. This allows for the regulation of cation density with the same number of cationic ligands, strengthening the interaction between the cationic ligands and the bacterial cell membrane, and improving antibacterial efficacy. The unique feature of this structure lies in the ability of the ring molecules to perform controllable "sliding" and "rotational" molecular motions under external stimuli. By using epichlorohydrin to lock the rings, the molecular mobility of the ring molecules can be adjusted. As the degree of ring locking gradually increases, the degree of freedom of the ring molecules is somewhat restricted, and their molecular mobility decreases accordingly. However, with increasing ring locking, the ring molecules are covalently linked together, reducing the distance between them. This makes the previously dispersed distribution of cationic ligands more concentrated, significantly enhancing the synergistic effect between the cationic ligands. This dynamically changing molecular structure allows polyrotaxane to interact more flexibly with bacterial cell membranes. By regulating the balance between cyclic molecular mobility and cation cooperativity, it maximizes the contact between cationic ligands and bacteria, thereby improving antibacterial efficacy. Furthermore, with the same number of cationic ligands, this structure can further regulate the distribution of cationic ligands to achieve a higher cationic ligand density, thus enhancing antibacterial properties.

[0010] Furthermore, the average number of connecting structures (bridge bonds) on each polyrotaxane ring molecule in the cationic ring-locked polyrotaxane is 2 to 4.

[0011] Preferably, the average number of connecting structures (bridge bonds) on each polyrotaxane ring molecule in the cationic ring-locked polyrotaxane is 2.1 to 3.8.

[0012] Furthermore, the cationic ligand is a ligand that has cationic properties and can interact with negatively charged substances (such as bacterial cell membranes).

[0013] Furthermore, the cationic ligand is 2,3-epoxypropyltrimethylammonium chloride or pentaethylenehexamine.

[0014] Furthermore, the axial molecule is polyethylene glycol or a polyethylene glycol block copolymer with a molecular weight of 4000-35000 Da.

[0015] Furthermore, the polyethylene glycol block copolymer is Pluronic L64, Pluronic F127, or Pluronic F68.

[0016] Furthermore, the cyclic molecule is one of α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin. The bridging agent can lock the cyclic molecule by bridging the hydroxyl groups on the coaxial cyclic molecule.

[0017] Furthermore, the end-capping structure is formed by the reaction of adamantane with the terminal carboxyl group of the axial molecule.

[0018] The present invention also provides a method for preparing the above-mentioned cationic ring-locked polyrotaxane, wherein the preparation method comprises adding a bridging agent to the polyrotaxane system to obtain the ring-locked polyrotaxane, and then adding a cationic ligand to obtain the final product; wherein the bridging agent is one of epichlorohydrin or diisocyanate compounds.

[0019] Furthermore, the concentration of epichlorohydrin used is 0–0.508 mol / L (excluding the case of a concentration of 0, 0 < C ≤ 0.508 mol / L). Different concentrations of bridging agents can regulate the degree of ring-locking of polyrotaxanes. Within a given range of bridging agent concentrations, bridging only occurs between coaxial ring molecules, while non-coaxial ring molecules do not form bridging bonds. This is because coaxial ring molecules are closest to each other and these ring molecules are strung on the same axis. Their diffusion direction in solution is relatively fixed, mainly exhibiting axial sliding. Therefore, bridging is more likely to occur between coaxial cyclodextrin molecules. Only bridging between coaxial ring molecules can effectively regulate the degree of ring-locking of individual polyrotaxane molecules. When the bridging agent concentration exceeds this range, non-coaxial bridging occurs between cyclodextrin molecules, leading to cross-linking between polyrotaxane molecules. Cross-linking between three or more polyrotaxane molecules may even occur, thus failing to effectively control the movement of ring molecules on the polyrotaxane axis.

[0020] Furthermore, the method for adding epichlorohydrin involves dissolving polyrotaxane in a solution of sodium hydroxide, potassium hydroxide, or potassium tert-butoxide, and then adding epichlorohydrin. The role of sodium hydroxide is to deprotonate the hydroxyl groups on the polyrotaxane ring molecule, thereby increasing its nucleophilicity. Because polyrotaxane can dissolve in a 1 mol / L sodium hydroxide solution, acid will be generated during the reaction to neutralize the sodium hydroxide. The more bridging agent added, the more sodium hydroxide is consumed. Therefore, to ensure that the sodium hydroxide concentration in the reaction system is 1 mol / L after the reaction is complete, the amount of sodium hydroxide used will vary depending on the amount of bridging agent used to avoid precipitation.

[0021] Furthermore, the diisocyanate compound is terephthalic diisocyanate or o-phthalic diisocyanate.

[0022] Furthermore, the method for adding the terephthalic diisocyanate is to dissolve polyrotaxane in DMSO and then add the terephthalic diisocyanate.

[0023] As a preferred embodiment, the method for preparing the cationic polyrotaxane includes the following steps:

[0024] S1. Polyrotaxane was synthesized via a heterogeneous reaction using polyethylene glycol as the axial molecule, α-cyclodextrin as the cyclic molecule, and adamantane as the end-capping agent.

[0025] S2. Dissolve polyrotaxane in sodium hydroxide solution, slowly add epichlorohydrin as a bridging agent, lock the ring molecules by bridging the hydroxyl groups on the coaxial ring molecules to control the molecular motion and coordination of the polyrotaxane ring molecules. By changing the concentration of the bridging agent and the reaction conditions, a series of ring-locked polyrotaxanes with different degrees of ring locking can be obtained.

[0026] S3. Using 2,3-epoxypropyltrimethylammonium chloride as a cationic ligand, under alkaline conditions, the ligand is modified onto a series of polyrotaxanes with different ring-locking degrees to ensure that the average number of cationic ligands modified per ring on the polyrotaxane molecule is the same.

[0027] This invention explores the influence of the molecular motion characteristics of polyrotaxane and its synergistic effect with cationic ligands on the antibacterial properties of a series of polyrotaxane ring molecules with different molecular motility and synergistic properties. Antibacterial efficacy was characterized by antibacterial experiments, and blood compatibility was characterized by hemolysis experiments. Experimental results show that, compared with existing cationic antibacterial polymers, this invention utilizes the unique dynamic structure of polyrotaxane to enhance the dynamism and synergistic effect of cationic ligands, thereby enhancing its interaction with bacterial cell membranes. It maintains strong antibacterial activity without significantly increasing cation density, thus avoiding the cytotoxic side effects caused by excessively high cation density in traditional cationic polymers. Therefore, the cationic ring-locked polyrotaxane of this invention can achieve both strong antibacterial effects and low cytotoxicity at low cation density, providing a new strategy for bacterial treatment and possessing clinical application potential.

[0028] Therefore, the present invention also provides the use of the above-mentioned cationic ring-locked polyrotaxane in antibacterial applications or in the preparation of antibacterial products.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] This invention provides a cationic ring-locked polyrotaxane, wherein the polyrotaxane comprises an axial molecule, a ring molecule, a capped structure, and a linkage structure formed by the chemical reaction of coaxially adjacent ring molecules with hydroxyl groups on adjacent ring molecules; the axial molecule is polyethylene glycol or a polyethylene glycol block copolymer; the ring molecule is cyclodextrin; and the ring molecule is modified with cationic ligands. The results of this invention show that when the polyrotaxane ring molecule is partially ring-locked, a balance between the dynamics and synergistic effects of the cationic ligands on the ring molecule can be achieved, significantly improving the bactericidal ability of the cationic ring-locked polyrotaxane. This allows the cationic antimicrobial polymer to possess high antimicrobial efficacy under low cation density conditions without causing toxicity to normal cells, thus enhancing the ability of the cationic antimicrobial polymer to balance antimicrobial and cytotoxic properties. This invention aims to develop a cationic ring-locked polyrotaxane based on a polyrotaxane structure, which, through innovative molecular design, improves the bactericidal effect and has significant clinical application potential, promoting the development of antimicrobial strategies towards a more efficient and safer direction. Attached Figure Description

[0031] Figure 1 This is a schematic diagram of the synthesis of cationic polyrotaxane.

[0032] Figure 2 The 1H NMR spectrum of the cationic polyrotaxane CPR(0) prepared according to Example 1 ( 1 H NMR, D2O, 298K).

[0033] Figure 3 The 1H NMR spectrum of the cationic ring-locked polyrotaxane CPR (1.1) prepared according to Example 2 ( 1 HNMR, D2O, 298K).

[0034] Figure 4 The 1H NMR spectrum of the cationic ring-locked polyrotaxane CPR (2.1) prepared according to Example 3 ( 1 HNMR, D2O, 298K).

[0035] Figure 5 The 1H NMR spectrum of the cationic ring-locked polyrotaxane CPR (3.8) prepared according to Example 4 ( 1 HNMR, D2O, 298K).

[0036] Figure 6 The 1H NMR spectrum of the cationic ring-locked polyrotaxane CPR (5.9) prepared according to Example 5 ( 1 HNMR, D2O, 298K).

[0037] Figure 7 Zeta potential diagrams of cationic ring-locked polyrotaxanes prepared according to Examples 1-5.

[0038] Figure 8 The figure shows the antibacterial test results of the cationic ring-locked polyrotaxanes prepared according to Examples 1-5.

[0039] Figure 9 The figure shows the hemolysis test results of the cationic ring-locked polyrotaxanes prepared according to Examples 1-5.

[0040] Figure 10 The figure shows the cytotoxicity test results of the cationic ring-locked polyrotaxanes prepared according to Examples 1-5. Detailed Implementation

[0041] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0042] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0043] Example 1 Synthesis of cationic polyrotaxane

[0044] (1) Synthesis of terminal carboxylated polyethylene glycol

[0045] Weigh 30 g of polyethylene glycol (35k-PEG, 30.0 g, 0.86 mmol, -OH 1.7 mmol) into a reaction flask, add 300 mL of pure water, and stir until the PEG is completely dissolved. Then add 2,2,6,6-tetramethylpiperidine oxide (TEMPO, 300 mg, 1.92 mmol) to the above solution and stir until completely dissolved. Subsequently, add sodium bromide (NaBr, 3.0 g, 30 mmol) and sodium hypochlorite (NaClO, 15 mL, available chlorine >5.0%) solutions sequentially, and stir the reaction at room temperature for 15 minutes. After the reaction is complete, add 10 mL of ethanol to quench the reaction, and then adjust the pH to <2 with hydrochloric acid solution. Extract the reaction solution with dichloromethane, collect the lower organic phase, concentrate it by rotary evaporation, slowly pour the concentrate into diethyl ether to precipitate, collect the precipitate by filtration, and dry it under vacuum to obtain terminal carboxylated polyethylene glycol 35k-PEG-(COOH)2.

[0046] (2) Synthesis of polyrotaxane

[0047] Terminally carboxylated polyethylene glycol (35k-PEG-(COOH)2, 12.0 g, 0.34 mmol) and α-cyclodextrin (α-CD, 48.0 g, 49.4 mmol) were dissolved separately in deionized water, with 35k-PEG-(COOH)2 dissolved in 60 mL of pure water and α-CD dissolved in 300 mL of pure water (heating in a 60°C water bath can promote dissolution). Subsequently, the 35k-PEG-(COOH)2 aqueous solution was slowly added dropwise to the α-CD aqueous solution, with the system being stirred uniformly during the addition. After the addition was complete, the reaction system was continuously stirred at 60°C for 6 hours, allowing the hydrophobic segments of 35k-PEG-(COOH)2 and the cavity of α-CD to self-assemble into a quasi-polyrotaxane structure through host-guest interactions. After the reaction was completed, the resulting white paste was freeze-dried to remove the solvent, yielding the quasi-polyrotaxane (PPR) intermediate product.

[0048] Next, quasi-polyrotaxane (PPR, 15.0 g, 0.09 mmol), 1-adamantaneamine (Ad-NH2, 1.3 g, 8.6 mmol), and Carter's condensing agent (BOP, 0.38 g, 0.86 mmol) were weighed into a reaction vessel and thoroughly mixed by solid shaking or mechanical stirring. Then, approximately 5 mL of anhydrous N,N-dimethylformamide (DMF) was slowly added dropwise to wet the reaction system, followed by dropwise addition of N,N-diisopropylethylamine (DIPEA, 0.15 mL, 0.86 mmol) dispersed in 1 mL of anhydrous DMF. The addition of anhydrous DMF continued until a thick slurry was formed. The amount of DMF used was strictly controlled and the volume added was recorded to ensure the reaction system remained heterogeneous. The reaction mixture was stirred continuously overnight at 4°C to allow the terminal carboxyl groups of 1-adamantaneamine and quasi-polyrotaxane to undergo an amidation reaction catalyzed by BOP and DIPEA. After the reaction was complete, the product was washed twice with a DMF / methanol (1:1, v / v) mixture and twice with methanol, with stirring for 30 minutes each time. The washed product was dissolved in an appropriate amount of DMSO and then added to water for recrystallization. The precipitate was collected by centrifugation (10,000 rpm, 10 minutes), and then washed with excess water to remove excess solvent and impurities. The resulting grayish-white solid was freeze-dried to obtain adamantane-terminated polyrotaxane (PR).

[0049] (3) Synthesis of cationic polyrotaxane (CPR)

[0050] Polyrotaxane (50 mg, 0.0003664 mmol) was dissolved in 1 mL of 1 mol / L sodium hydroxide solution and stirred until completely dissolved. Then, 2,3-epoxypropyltrimethylammonium chloride (cationic ligand, 832 mg, 5.4872 mmol, CAS#:3033-77-0) was dissolved in 1 mL of 1 mol / L sodium hydroxide solution, and this solution was slowly added dropwise to the sodium hydroxide solution containing polyrotaxane. The reaction was carried out overnight at room temperature. After the reaction was complete, the product was dialyzed with pure water until the dialysate was neutral. The dialyzed product was lyophilized to obtain a crude product. The lyophilized crude product was dissolved in an appropriate amount of anhydrous dichloromethane and stirred at room temperature for 24 hours. A white solid product was obtained by filtration and then vacuum dried at room temperature for 24 hours to obtain the final product, denoted as CPR(0), i.e., non-locked ring with an average of 0 molecular bridge bonds per ring.

[0051] A schematic diagram of the synthesis of cationic polyrotaxane is shown below. Figure 1 As shown, the axon molecule passes through the cavity of the ring molecule through host-guest interactions, and the ends of the axon molecule are sealed with sterically hindered groups to prevent the ring molecule from sliding out. The ring molecule on the polyrotaxane axis can slide and rotate along the axis. The 1H NMR spectrum of the cationic polyrotaxane CPR(0) synthesized by this method is shown. 1 H NMR, D2O, 298K) such as Figure 2 As shown, the characteristic peaks of the cationic ligands at 4.4 ppm and 3.25 ppm confirm the successful modification of the cationic ligands. The average ring-penetrating number of the cationic polyrotaxane was calculated to be 105, the ring-penetrating rate was 26.5%, and the average number of cationic ligands per ring was 4.9.

[0052] Example 2 Synthesis of cationic ring-locked polyrotaxane

[0053] The method for synthesizing polyrotaxane (PR) is as described in steps (1) and (2) of Example 1.

[0054] (3) Synthesis of ring-locked polyrotaxanes

[0055] The end-capped polyrotaxane was dissolved in NaOH aqueous solution (1.01 mol / L, 3 mL). After complete dissolution, the reaction solution was heated to 60 °C, and epichlorohydrin (EPI, 0.0635 mol / L, 15 μL) was slowly added dropwise. The reaction was carried out at 60 °C for 24 hours. After the reaction was completed, the reaction solution was cooled to room temperature and dialyzed to remove small molecule impurities. The dialysate was centrifuged to separate the white precipitate, which was then lyophilized to obtain the ring-locked polyrotaxane, denoted as PR(1.1).

[0056] (4) Synthesis of cationic ring-locked polyrotaxane

[0057] The above-mentioned cyclic polyrotaxane (51 mg, 0.0003664 mmol) was dissolved in 1 mL of 1 mol / L sodium hydroxide solution and stirred until completely dissolved. Then, 2,3-epoxypropyltrimethylammonium chloride (809 mg, 5.3344 mmol) was dissolved in 1 mL of 1 mol / L sodium hydroxide solution, and this solution was slowly added dropwise to the sodium hydroxide solution containing the cyclic polyrotaxane. The reaction was carried out overnight at room temperature. After the reaction was complete, the product was dialyzed with pure water until the dialysate was neutral. The dialyzed product was lyophilized to obtain a crude product. The lyophilized crude product was washed with an appropriate amount of anhydrous dichloromethane, stirred at room temperature for 24 hours, and separated by vacuum filtration to obtain a white solid product. This solid product was then vacuum dried at room temperature for 24 hours to obtain the final product, denoted as CPR(1.1), which represents an average of 1.1 bridging bonds per ring molecule.

[0058] The proton NMR spectrum of the cationic ring-locked polyrotaxane CPR(1.1) synthesized by this method ( 1 H NMR, D2O, 298K) such as Figure 3 As shown, the characteristic peaks of the cationic ligands at 4.4 ppm and 3.25 ppm confirm the successful modification of the cationic ligands. The average ring-penetrating number of the cationic polyrotaxane was calculated to be 105, the ring-penetrating rate was 26.5%, and the average number of cationic ligands per ring was 4.9.

[0059] Example 3 Synthesis of cationic ring-locked polyrotaxane

[0060] The method for synthesizing polyrotaxane (PR) is as described in steps (1) and (2) of Example 1.

[0061] (3) Synthesis of ring-locked polyrotaxanes

[0062] The end-capped polyrotaxane was dissolved in NaOH aqueous solution (1.03 mol / L, 3 mL). After complete dissolution, the reaction solution was heated to 60 °C, and epichlorohydrin (EPI, 0.127 mol / L, 30 μL) was slowly added dropwise. The reaction was carried out at 60 °C for 24 hours. After the reaction was completed, the reaction solution was cooled to room temperature and dialyzed to remove small molecule impurities. The dialysate was centrifuged to separate the white precipitate, which was then lyophilized to obtain the ring-locked polyrotaxane, denoted as PR(2.1), indicating an average of 2.1 bridging bonds per ring molecule.

[0063] (4) Synthesis of cationic ring-locked polyrotaxane

[0064] The above-mentioned cyclic polyrotaxane (52.6 mg, 0.0003664 mmol) was dissolved in 1 mL of 1 mol / L sodium hydroxide solution and stirred until completely dissolved. Then, 2,3-epoxypropyltrimethylammonium chloride (789 mg, 5.2056 mmol) was dissolved in 1 mL of 1 mol / L sodium hydroxide solution, and this solution was slowly added dropwise to the sodium hydroxide solution containing the cyclic polyrotaxane. The reaction was carried out overnight at room temperature. After the reaction was complete, the product was dialyzed with pure water until the dialysate was neutral. The dialyzed product was lyophilized to obtain a crude product. The lyophilized crude product was washed with an appropriate amount of anhydrous dichloromethane, stirred at room temperature for 24 hours, and separated by vacuum filtration to obtain a white solid product. This solid product was then vacuum dried at room temperature for 24 hours to obtain the final product, denoted as CPR (2.1).

[0065] The proton NMR spectrum of the cationic ring-locked polyrotaxane CPR (2.1) synthesized by this method ( 1 H NMR, D2O, 298K) such as Figure 4 As shown, the characteristic peaks of the cationic ligands at 4.4 ppm and 3.25 ppm confirm the successful modification of the cationic ligands. The average ring-penetrating number of the cationic polyrotaxane was calculated to be 105, the ring-penetrating rate was 26.5%, and the average number of cationic ligands per ring was 4.9.

[0066] Example 4 Synthesis of cationic ring-locked polyrotaxane

[0067] The method for synthesizing polyrotaxane (PR) is as described in steps (1) and (2) of Example 1.

[0068] (3) Synthesis of ring-locked polyrotaxanes

[0069] The end-capped polyrotaxane was dissolved in a NaOH aqueous solution (1.05 mol / L, 3 mL). After complete dissolution, the reaction solution was heated to 60 °C, and epichlorohydrin (EPI, 0.254 mol / L, 60 μL) was slowly added dropwise. The reaction was carried out at 60 °C for 24 hours. After the reaction was completed, the reaction solution was cooled to room temperature and dialyzed to remove small molecule impurities. Subsequently, the product was lyophilized to obtain a crude product. The lyophilized crude product was washed with an appropriate amount of anhydrous dichloromethane and stirred at room temperature for 24 hours. A white solid product was obtained by filtration and then vacuum dried at room temperature for 24 hours to obtain the ring-locked polyrotaxane, denoted as PR(3.8), which means that the average number of bridging bonds per ring molecule is 3.8.

[0070] (4) Synthesis of cationic ring-locked polyrotaxane

[0071] The above-mentioned cyclic polyrotaxane (54.6 mg, 0.0003664 mmol) was dissolved in 1 mL of 1 mol / L sodium hydroxide solution and stirred until completely dissolved. Then, 2,3-epoxypropyltrimethylammonium chloride (755 mg, 4.98 mmol) was dissolved in 1 mL of 1 mol / L sodium hydroxide solution, and this solution was slowly added dropwise to the sodium hydroxide solution containing the cyclic polyrotaxane. The reaction was carried out overnight at room temperature. After the reaction was complete, the product was dialyzed with pure water until the dialysate was neutral. The dialyzed product was lyophilized to obtain a crude product. The lyophilized crude product was washed with an appropriate amount of anhydrous dichloromethane, stirred at room temperature for 24 hours, and separated by vacuum filtration to obtain a white solid product. This solid product was then vacuum dried at room temperature for 24 hours to obtain the final product, denoted as CPR (3.8).

[0072] The proton NMR spectrum of the cationic ring-locked polyrotaxane CPR(3.8) synthesized by this method ( 1 H NMR, D2O, 298K) such as Figure 5 As shown, the characteristic peaks of the cationic ligands at 4.4 ppm and 3.25 ppm confirm the successful modification of the cationic ligands. The average ring-penetrating number of the cationic polyrotaxane was calculated to be 105, the ring-penetrating rate was 26.5%, and the average number of cationic ligands per ring was 4.9.

[0073] Example 5 Synthesis of cationic ring-locked polyrotaxane

[0074] The method for synthesizing polyrotaxane (PR) is as described in steps (1) and (2) of Example 1.

[0075] (3) Synthesis of ring-locked polyrotaxanes

[0076] The end-capped polyrotaxane was dissolved in a NaOH aqueous solution (1.1 mol / L, 3 mL). After complete dissolution, the reaction solution was heated to 60 °C, and epichlorohydrin (EPI, 0.508 mol / L, 119 μL) was slowly added dropwise. The reaction was carried out at 60 °C for 24 hours. After the reaction was completed, the reaction solution was cooled to room temperature and dialyzed to remove small molecule impurities. Subsequently, the product was lyophilized to obtain a crude product. The lyophilized crude product was washed with an appropriate amount of anhydrous dichloromethane and stirred at room temperature for 24 hours. A white solid product was obtained by filtration and then vacuum dried at room temperature for 24 hours to obtain the ring-locked polyrotaxane, denoted as PR(5.9), which means that the average number of bridging bonds per ring molecule is 5.9.

[0077] (4) Synthesis of cationic ring-locked polyrotaxane

[0078] The above-mentioned cyclic polyrotaxane (57.4 mg, 0.0003664 mmol) was dissolved in 1 mL of 1 mol / L sodium hydroxide solution and stirred until completely dissolved. Then, 2,3-epoxypropyltrimethylammonium chloride (710 mg, 4.68 mmol) was dissolved in 1 mL of 1 mol / L sodium hydroxide solution, and this solution was slowly added dropwise to the sodium hydroxide solution containing the cyclic polyrotaxane. The reaction was carried out overnight at room temperature. After the reaction was complete, the product was dialyzed with pure water until the dialysate was neutral. The dialyzed product was lyophilized to obtain a crude product. The lyophilized crude product was washed with an appropriate amount of anhydrous dichloromethane, stirred at room temperature for 24 hours, and separated by vacuum filtration to obtain a white solid product. This solid product was then vacuum dried at room temperature for 24 hours to obtain the final product, denoted as CPR (5.9).

[0079] The proton NMR spectrum of the cationic ring-locked polyrotaxane CPR(5.9) synthesized by this method ( 1 H NMR, D2O, 298K) such as Figure 6 As shown, the characteristic peaks of the cationic ligands at 4.4 ppm and 3.25 ppm confirm the successful modification of the cationic ligands. The average ring-penetrating number of the cationic polyrotaxane was calculated to be 105, the ring-penetrating rate was 26.5%, and the average number of cationic ligands per ring was 4.9.

[0080] Through reasonable structural design and reaction control, a series of polyrotaxanes with different ring-locking degrees were successfully synthesized. Furthermore, through ligand modification reactions, cationic ligands were successfully modified onto the polyrotaxane ring molecules, ensuring that the average number of cationic ligands per ring was consistent for each group of polyrotaxanes.

[0081] Zeta potential data for cationic polyrotaxanes prepared in Examples 1-5 are as follows: Figure 7 As shown in the figure, it is evident that even though the average number of cationic ligands per ring is the same for each group of polyrotaxane samples, the zeta potential gradually increases with the increase of the number of locked rings. This phenomenon can be explained as follows: under the condition that the ratio of axis, ring, and ligand in polyrotaxane is the same, the bridging structure restricts the spatial arrangement of ring molecules. The bridging structure connects the originally relatively independent ring molecules together through covalent bonds, making the ring molecules more compact, thus leading to a more concentrated distribution of cationic ligands on the ring and an increased cation density. In contrast, in unbridged polyrotaxane, each ring molecule remains relatively independent, and the distribution of cationic ligands is more dispersed. This results in a lower cation density on each ring molecule, leading to a lower zeta potential. Therefore, the zeta potential increases with the increase of the number of bridging bonds.

[0082] Example 6: Antibacterial effect of cationic ring-locked polyrotaxane

[0083] To investigate the effect of the ring-locking degree of polyrotaxane on its antibacterial properties, cationic polyrotaxanes with different ring-locking degrees were prepared into solutions with the same cationic ligand concentration (0.25 μg / mL) and co-cultured with *E. coli* at 37℃ and 125 rpm for 4 hours. After incubation, bacterial viability was calculated using the bacterial plating method: bacterial viability (%) = (number of bacteria in the experimental group / number of bacteria in the control group) * 100%. Lower bacterial viability after 4 hours of co-culture indicated stronger antibacterial effects.

[0084] The antibacterial effects of the cationic polyrotaxanes prepared in Examples 1-5 at a concentration of 0.25 μg / mL are as follows: Figure 8 As shown, experimental results indicate that by controlling the movement of ring molecules with appropriate inter-ring bridging agents, the coordinated aggregation of cationic ligands on the ring molecules can be achieved within a certain region. This balance between the dynamics and synergy of cationic ligands on the ring molecules can be realized, thereby enhancing the interaction between cationic ligands and the cell membrane to achieve optimal antibacterial effects. When the average number of bridging bonds per ring molecule in cationic polyrotaxane is between 2.1 and 3.8, the antibacterial effect of cationic polyrotaxane is significantly enhanced at the same cationic ligand concentration. Therefore, the dynamic balance between molecular dynamics and cationic synergy of cationic polyrotaxane can be achieved by bridging coaxial ring molecules to achieve the best antibacterial effect.

[0085] Example 7 Hemolysis test of cationic ring-locked polyrotaxane

[0086] To evaluate the blood compatibility of the cationic polyrotaxanes prepared in Examples 1-5, the hemolysis rate was used to characterize the material's effect on erythrocytes. The hemolysis rate refers to the proportion of erythrocytes that dissolve upon contact with the material; a hemolysis rate exceeding 5% is considered to indicate toxicity and potential adverse effects on blood or host tissues. This experiment allows for a preliminary assessment of the safety of cationic polyrotaxanes in vivo, particularly their reaction upon contact with blood.

[0087] The results of blood compatibility are as follows Figure 9 As shown, no significant hemolysis was observed in each group of cationic polyrotaxanes within the cationic ligand concentration range of 0–64 μg / mL, and the hemolysis rate was within 5%, which meets the requirements for blood safety.

[0088] Example 8 Cytotoxicity test of cationic ring-locked polyrotaxane

[0089] To characterize the cytotoxicity of the material, mouse embryonic fibroblasts (NIH 3T3) were selected for evaluation. Cells were seeded in 96-well plates and pre-cultured in an incubator (37°C, 5% CO2) for 12-24 hours to allow the cells to reach the exponential phase. The culture medium was aspirated from each well, and the material with a cationic ligand concentration of 1 μg / mL was added to the culture plate for co-culturing with the cells for 24 hours. After 24 hours, cell viability was assessed using a CCK-8 assay kit according to the user manual, with fresh culture medium as a control. Five replicates were performed per group.

[0090] The results of cytotoxicity are as follows Figure 10 As shown, the cell viability of each group of materials after co-culture was greater than 80%, indicating that the materials did not produce significant toxicity to the cells and met the requirements for biosafety.

[0091] Example 9 Synthesis of Ring-locked Polyrotaxane

[0092] The method for synthesizing polyrotaxane (PR) is as described in steps (1) and (2) of Example 1.

[0093] 1 g of polyrotaxane was dissolved in 30 mL of anhydrous dimethyl sulfoxide (DMSO). Under nitrogen protection, dibutyltin dilaurate (DBTDL, 20 μL) was added sequentially as a catalyst, and 2,6-di-tert-butyl-p-cresol (BHT, 1.0 mg, 0.0047 mmol) pre-dissolved in 3 mL of anhydrous DMSO. Subsequently, terephthalic diisocyanate (62 mg, 160 g / mol) dissolved in 3 mL of anhydrous DMSO was slowly added dropwise. After stirring until homogeneous, the reaction was carried out at 40 °C for 12 h. After the reaction was completed, the resulting reaction solution was slowly poured into excess ice-cold acetone to precipitate the product. The precipitate was collected by centrifugation. The precipitate was washed with acetone and then dried under vacuum to obtain the final product.

[0094] Experimental results show that terephthalic diisocyanate can also be used as a bridging agent to adjust the ring-locking degree of polyrotaxane molecules. Moreover, after modification with cationic ligands, the antibacterial effect of cationic polyrotaxane is significantly enhanced under the same cationic ligand concentration.

[0095] Example 10 Synthesis of cationic ring-locked polyrotaxane

[0096] The method for synthesizing ring-locked polyrotaxane (PR) is as described in steps (1), (2) and (3) of Examples 2-5.

[0097] (4) 151 mg of N,N'-carbonyldiimidazole (CDI) was dissolved in 5 mL of anhydrous DMSO. Separately, 100 mg of cyclic polyrotaxane was dissolved in 10 mL of anhydrous DMSO. The polyrotaxane solution was then slowly added dropwise to the CDI solution under nitrogen protection, and the reaction was stirred at room temperature for 16 h. After the reaction, the resulting solution was precipitated using a mixed solvent of tetrahydrofuran (THF) and diethyl ether (Et2O) in a 1:2 volume ratio. The precipitate was collected by centrifugation and washed three times with THF. The resulting wet solid was dissolved in 10 mL of anhydrous DMSO, and then slowly added dropwise to 303 μL of pentaethylenehexamine (PEHA) solution diluted in 5 mL of anhydrous DMSO. The reaction was stirred at room temperature for 20 h. After the reaction, the product was precipitated with THF. The precipitate was collected by centrifugation, dissolved in an appropriate amount of water, and dialyzed for 3 days. Finally, the target product was obtained by freeze-drying.

[0098] Experimental results show that, under the same cationic ligand concentration, the antibacterial effect of the cyclic polyrotaxane modified with pentaethylenehexamine is significantly enhanced.

Claims

1. A cationic ring-locked polyrotaxane, characterized in that, The polyrotaxane comprises an axial molecule, a ring molecule, a capped structure, and a connecting structure formed by a bridging agent reacting chemically with the hydroxyl groups on the adjacent ring molecules; the axial molecule is polyethylene glycol or a polyethylene glycol block copolymer; the ring molecule is cyclodextrin; the ring molecule is modified with a cationic ligand; the bridging agent is one of epichlorohydrin or a diisocyanate compound; the concentration of epichlorohydrin used is 0 < C ≤ 0.508 mol / L; the cationic ligand is 2,3-epoxypropyltrimethylammonium chloride or pentaethylenehexamine.

2. The cationic ring-locked polyrotaxane according to claim 1, characterized in that, The average number of linking structures on each polyrotaxane ring molecule in the cationic ring-locked polyrotaxane is 2 to 4.

3. The cationic ring-locked polyrotaxane according to claim 1, characterized in that, The axial molecules are polyethylene glycol or polyethylene glycol block copolymers with a molecular weight of 4000 to 35000 Da.

4. The cationic ring-locked polyrotaxane according to claim 1, characterized in that, The cyclic molecule is one of α-cyclodextrin, β-cyclodextrin, or γ-cyclodextrin.

5. The cationic ring-locked polyrotaxane according to claim 1, characterized in that, The end-capped structure is formed by the reaction of adamantane with the terminal carboxyl group of the axial molecule.

6. The method for preparing the cationic ring-locked polyrotaxane according to any one of claims 1 to 5, characterized in that, A bridging agent is added to the polyrotaxane system to obtain a ring-locked polyrotaxane, and then a cationic ligand is added to obtain the final product; the bridging agent is one of epichlorohydrin or a diisocyanate compound; the concentration of epichlorohydrin used is 0 < C ≤ 0.508 mol / L.

7. The method according to claim 6, characterized in that, The method for adding epichlorohydrin is to dissolve polyrotaxane in any one of sodium hydroxide, potassium hydroxide or potassium tert-butoxide solutions, and then add epichlorohydrin.

8. The method according to claim 6, characterized in that, The diisocyanate compounds are terephthalic diisocyanate and o-phthalic diisocyanate.

9. The use of the cationic ring-locked polyrotaxane according to any one of claims 1 to 5 in the preparation of antibacterial products.

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