Preparation method and application of cationic polyrotaxane

Abstract

The invention discloses cationized ring-locked polyrotaxane. The polyrotaxane is composed of an axial molecule, a ring molecule, an end-capping structure and a connecting structure, wherein the connecting structure is formed between coaxially adjacent ring molecules through a chemical reaction with hydroxyl groups on the adjacent ring molecules; the axial molecule is polyethylene glycol or a polyethylene glycol block copolymer; the cyclic molecules are cyclodextrin; a cation ligand is modified on the ring molecule. Research results show that when part of polyrotaxane ring molecules are locked, balance of dynamic property and synergism of cationic ligands on the ring molecules can be realized, and the bactericidal ability of cationized ring-locked polyrotaxane is remarkably improved, so that the cationic antibacterial polymer has a relatively high antibacterial effect under the condition of low cationic density, and the cationic antibacterial polymer can be used for preparing antibacterial agents. And no toxic effect is generated on normal cells. The invention aims to develop the cationized locked-ring polyrotaxane based on the polyrotaxane structure, through innovative molecular design, the effect of killing bacteria is improved, and the cationized locked-ring polyrotaxane has remarkable clinical application potential.

Description

Technical Field

[0001] The present invention relates to the field of biomedical materials, and in particular to a preparation method and application of a cationic polyrotaxane. Background Art

[0002] In the intersection of modern materials science and biomedicine, how to effectively and safely fight bacterial infections has always been an important topic of concern. The bacterial cell membrane is a key target in the treatment of drug-resistant bacteria. Its structure is relatively stable and not prone to mutation. It has become an important direction for the study of antibacterial strategies in recent years. In particular, the electronegative characteristics of the bacterial cell membrane provide a unique entry point for antibacterial therapy. The negative charge on the bacterial cell membrane makes the membrane strongly attract positively charged molecules. Therefore, cationic antibacterial 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, destroying the integrity of the cell membrane, thereby inhibiting or killing bacteria. In order to improve the antibacterial properties of cationic antibacterial polymers, cationic polymers are usually required to have a higher cation density, but high cation density is often accompanied by higher cytotoxicity, and even leads to overreaction of the immune system, causing serious toxic side effects. Therefore, how to reduce toxicity to host cells while enhancing the antibacterial effect is a key issue in the study of cationic antibacterial polymers.

[0003] As a typical representative of artificial molecular machines, polyrotaxanes provide new ideas for solving this problem with their unique molecular structure. Polyrotaxanes are composed of an axis molecule and multiple macrocyclic molecules. The axis molecule passes through the cavity of the macrocyclic molecule, and the ring molecule is connected to the axis molecule by mechanical interlocking. The two ends of the macrocyclic molecule are blocked by a large capping agent to prevent the ring molecule from slipping out, thus forming a unique mechanical structure. The dynamic structure of polyrotaxanes gives it significant advantages in antibacterial treatment. Unlike traditional static cationic polymers, this dynamically changing molecular structure enables polyrotaxanes to interact with bacterial cell membranes more flexibly. However, at present, if we want to further improve the antibacterial properties of polyrotaxanes, we can only increase the density of cationic ligands by excessively increasing the number of cationic ligands, thereby improving the antibacterial properties of polyrotaxanes. Patent CN114949253A discloses a dual-drug-linked polyrotaxane nano-drug delivery system and its preparation method and application. It discloses that a cationic polyrotaxane nano-material (mPDA@PR-PHEA) with mesoporous polydopamine (mPDA) as the core is modified on its surface with a cationic polyrotaxane (PR-PHEA) with molecular mobility to obtain a clotrimazole and NO dual-drug linked polyrotaxane nano-drug delivery system. This dual-drug linked polyrotaxane nano-drug delivery system can effectively accelerate and enhance the full contact between the NO drug delivery system and the fungus, enhance the interaction between the material itself and the fungus, and on this basis improve the bioavailability of NO and the loaded drug, further realize the synergistic combination of NO and the drug, and has a therapeutic effect on Candida albicans and other related diseases. However, the patent only discloses how to use the polyrotaxane nano-drug delivery system to improve the bioavailability and mutual coordination of NO and loaded drugs to achieve better antibacterial effects, rather than regulating the mobility of the polyrotaxane ring molecules themselves to achieve a balance between the dynamics and synergy of the modified cationic ligands, thereby making the cationic polyrotaxane itself have better antibacterial properties and less cytotoxicity. Therefore, it is of great significance to provide a highly efficient and low-toxic cationic antibacterial polymer to promote the development of antibacterial strategies in a more efficient and safe direction. Summary of the invention

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

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

[0006] The 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 object of the present invention is achieved through the following technical solutions:

[0008] The present invention provides a cationic locked-ring polyrotaxane, which comprises an axis molecule, a ring molecule, a capping structure, and a connection structure formed by chemical reaction between coaxially adjacent ring molecules and hydroxyl groups on adjacent ring molecules; the axis 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] The present invention synthesizes polyrotaxane with an axis molecule, a ring molecule, and a capping agent, controls the dynamics of the ring molecule with a bridging agent, and then modifies the cationic ligand on the locked ring polyrotaxane. By utilizing the dynamics of the polyrotaxane ring molecule, changes are made on the carrier structure of the cationic ligand to enhance the dynamics and synergy of the cationic ligand. The regulation of the cationic density can be achieved under the same number of cationic ligands, and the interaction between the cationic ligand and the bacterial cell membrane is enhanced, thereby improving the antibacterial effect. The particularity of this structure is that the ring molecule can perform controllable "sliding" and "rotating" molecular motion under external stimulation. By using epichlorohydrin to lock the ring, the molecular motion ability of the ring molecule can be adjusted. When the degree of locking the ring gradually increases, the freedom of movement of the ring molecule will be subject to certain restrictions, and the molecular motion ability will decrease accordingly. However, with the increase of the degree of locking the ring, since the locking ring makes the ring molecules connected together by covalent bonds, the distance between the ring molecules is shortened after the locking ring, which makes the originally dispersed cationic ligand distribution more concentrated, and the synergy between the cationic ligands will be significantly enhanced. This dynamically changing molecular structure enables polyrotaxanes to interact with bacterial cell membranes more flexibly, and can maximize the contact between cationic ligands and bacteria by adjusting the balance between the motility of the ring molecules and the synergy of the cations, thereby improving the antibacterial effect. At the same time, with the same number of cationic ligands, this structure can also further regulate the distribution of cationic ligands to achieve a higher density of cationic ligands, thereby improving antibacterial performance.

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

[0011] Preferably, the average number of connecting structures (number of bridging 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 having cationic properties and capable of interacting with negatively charged substances (such as bacterial cell membranes).

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

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

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

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

[0017] Furthermore, the end-capping structure is formed by the reaction of adamantane amine or 2,4-dinitrofluorobenzene with the terminal carboxyl group of the axle molecule.

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

[0019] Furthermore, the epichlorohydrin is used at a concentration of 0 to 0.508 mol / L (excluding the case where the concentration is 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 occurs only between coaxial ring molecules, while bridging bonds do not form between non-coaxial ring molecules. This is because the distance between coaxial ring molecules is the shortest and these ring molecules are threaded on the same axis, and their diffusion direction in the solution is relatively fixed, mainly manifested as axial sliding, so coaxial cyclodextrin molecules are more likely to be bridged. Only the bridging between coaxial ring molecules can effectively regulate the degree of ring locking of ring molecules on a single polyrotaxane molecule. When the concentration of the bridging agent exceeds this range, non-coaxial bridging will occur between cyclodextrin molecules, resulting in cross-linking between polyrotaxane molecules, and even cross-linking between three or more polyrotaxane molecules, thereby failing to effectively control the movement of ring molecules on the polyrotaxane axis.

[0020] Furthermore, the method for adding epichlorohydrin is to dissolve the polyrotaxane in any solution of sodium hydroxide, potassium hydroxide or potassium tert-butoxide, and then add epichlorohydrin. The role of sodium hydroxide is to deprotonate the hydroxyl groups on the polyrotaxane ring molecules and improve nucleophilicity. Because the polyrotaxane can be dissolved in a 1 mol / L sodium hydroxide solution, acid will be generated during the reaction to neutralize the sodium hydroxide. The more the bridging agent is fed, the more sodium hydroxide is consumed. Therefore, in order to ensure that the sodium hydroxide concentration of the reaction system is 1 mol / L after the reaction is completed, the amount of sodium hydroxide used will vary according to the amount of the bridging agent used to avoid precipitation.

[0021] Furthermore, the diisocyanate compound is terephthalate diisocyanate or o-phthalate diisocyanate.

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

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

[0024] S1. Polyrotaxanes were synthesized by heterogeneous reaction using polyethylene glycol as the axis molecule, α-cyclodextrin as the ring molecule, and adamantane amine as the end-capping agent;

[0025] S2. dissolving the polyrotaxane in a sodium hydroxide solution, slowly dropping a bridging agent, epichlorohydrin, to lock the ring molecules by bridging the hydroxyl groups on the coaxial ring molecules to control the molecular motion and synergy 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 was modified on a series of polyrotaxanes with different degrees of ring locking to ensure that the average number of cationic ligands modified on each ring of the polyrotaxane molecule was the same.

[0027] The present invention explores the influence of the motion characteristics of polyrotaxane molecules and the synergy with cationic ligands on their antibacterial properties by modifying cationic ligands on a series of polyrotaxane ring molecules with different molecular motion capabilities and synergy. The antibacterial effect is characterized by antibacterial experiments, and the blood compatibility is characterized by hemolysis experiments. The experimental results show that compared with the existing cationic antibacterial polymers, the present invention utilizes the unique dynamic structure of polyrotaxane to enhance the dynamic and synergistic properties of cationic ligands, thereby enhancing its interaction with bacterial cell membranes, and can still maintain strong antibacterial activity without significantly increasing the cation density, thereby avoiding the cytotoxic side effects of traditional cationic polymers due to excessively high cation density. Therefore, the cationic locked ring polyrotaxane of the present invention can have both strong antibacterial effects and low cytotoxicity at low cation density, providing a new strategy for bacterial treatment and having 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] The present invention provides a cationic ring-locked polyrotaxane, which consists of an axis molecule, a ring molecule, a capping structure, and a connection structure formed by chemical reaction between coaxial adjacent ring molecules and hydroxyl groups on adjacent ring molecules; the axis 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. The research results of the present invention show that when the polyrotaxane ring molecule is partially ring-locked, the dynamic and synergistic balance of the cationic ligand on the ring molecule can be achieved, which significantly improves the bactericidal ability of the cationic ring-locked polyrotaxane, so that the cationic antibacterial polymer has a higher antibacterial effect under low cation density, and will not produce toxic effects on normal cells, and improves the ability of the cationic antibacterial polymer to balance antibacterial and cytotoxicity. The present invention aims to develop a cationic ring-locked polyrotaxane based on a polyrotaxane structure, which improves the effect of killing bacteria through innovative molecular design, has significant clinical application potential, and promotes the development of antibacterial strategies in a more efficient and safe direction. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Schematic diagram of the synthesis of cationic polyrotaxane.

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

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

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

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

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

[0037] Figure 7 : Zeta potential diagram of the cationic ring-locked polyrotaxane prepared according to Examples 1-5.

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

[0039] Fig. 9 Graph showing the results of a hemolysis experiment of a cationic ring-locked polyrotaxane prepared according to Examples 1-5.

[0040] Fig.10 The figure is a graph showing the cytotoxicity test results of the cationic ring-locked polyrotaxane prepared according to Examples 1-5. DETAILED DESCRIPTION

[0041] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

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

[0043] Example 1 Synthesis of cationic polyrotaxane

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

[0045] Weigh 30g of polyethylene glycol (35k-PEG, 30.0g, 0.86mmol, -OH 1.7mmol) in a reaction bottle, add 300mL of pure water, stir until PEG is completely dissolved, add 2,2,6,6-tetramethylpiperidinoxide (TEMPO, 300mg, 1.92mmol) to the above solution and stir until completely dissolved. Then add sodium bromide (NaBr, 3.0g, 30mmol)) and sodium hypochlorite (NaClO, 15mL, effective chlorine>5.0%) solution in sequence, stir and react at room temperature for 15 minutes. After the reaction is completed, add 10mL of ethanol to quench the reaction, and then adjust the pH to <2 with hydrochloric acid solution. The reaction solution is extracted with dichloromethane, the lower organic phase is taken, and after rotary evaporation and concentration, the concentrate is slowly poured into ether for precipitation, the precipitate is collected by suction filtration, and the terminal carboxyl polyethylene glycol 35k-PEG-(COOH)2 is obtained after vacuum drying.

[0046] (2) Polyrotaxane synthesis

[0047] Dissolve terminal carboxyl polyethylene glycol (35k-PEG-(COOH)2, 12.0 g, 0.34 mmol) and α-cyclodextrin (α-CD, 48.0 g, 49.4 mmol) in deionized water, respectively, wherein 35k-PEG-(COOH)2 is dissolved in 60 mL of pure water, and α-CD is dissolved in 300 mL of pure water (heated in a 60°C water bath to promote dissolution). Subsequently, the 35k-PEG-(COOH)2 aqueous solution is slowly added dropwise to the α-CD aqueous solution, and the system is stirred evenly during the addition. After the addition is completed, the reaction system is stirred continuously at 60°C for 6 hours to allow the hydrophobic segments of 35k-PEG-(COOH)2 and the cavities of α-CD to self-assemble to form a pseudopolyrotaxane structure through host-guest interactions. After the reaction is completed, the generated white paste is freeze-dried, and the solvent is removed to obtain a pseudopolyrotaxane (PPR) intermediate product.

[0048] Next, weigh pseudopolyrotaxane (PPR, 15.0 g, 0.09 mmol), 1-adamantanamine (Ad-NH2, 1.3 g, 8.6 mmol) and Carter condensation agent (BOP, 0.38 g, 0.86 mmol) in a reaction vessel and mix them thoroughly by solid shaking or mechanical stirring. Subsequently, about 5 mL of anhydrous N,N-dimethylformamide (DMF) was slowly added to the mixture to wet the reaction system, and then N,N-diisopropylethylamine (DIPEA, 0.15 mL, 0.86 mmol) solution dispersed with 1 mL of anhydrous DMF was added dropwise, and anhydrous DMF was continued to be added dropwise until the system formed a thick mud. During the process, the amount of DMF was strictly controlled and the drop volume was recorded to ensure that the reaction system was in a heterogeneous state. The reaction mixture was continuously stirred overnight at 4 ° C to allow 1-adamantanamine and the terminal carboxyl groups of the pseudopolyrotaxane to undergo amidation reaction under the catalysis of BOP and DIPEA. After the reaction was completed, the mixture was washed twice with a DMF / methanol (1:1, v / v) mixed solution and twice with methanol, and stirred for 30 minutes each time. The washed product was dissolved in an appropriate amount of DMSO and added to water for reprecipitation. The product precipitate was collected by centrifugation (10000 rpm, 10 minutes), and then washed with excess water to remove excess solvent and impurities. The resulting off-white solid was freeze-dried to obtain a polyrotaxane (PR) capped with adamantaneamine.

[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 it was 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 the solution was slowly added dropwise to the sodium hydroxide solution containing polyrotaxane, and the reaction was allowed to proceed overnight at room temperature. After the reaction, the product was dialyzed with pure water until the dialyzate 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, stirred at room temperature for 24 hours, and a white solid product was separated by suction filtration, and vacuum dried at room temperature for 24 hours to obtain the final product, which was recorded as CPR (0), i.e., no locked ring, and the average number of bridging bonds per ring molecule was 0.

[0051] The synthesis diagram of cationic polyrotaxane is shown in Figure 1 As shown in the figure, the axis molecule passes through the cavity of the ring molecule through the host-guest interaction, and the end of the axis molecule is capped with a large steric group to prevent the ring molecule from sliding out. The ring molecule on the axis of the polyrotaxane can slide and rotate along the axis. The nuclear magnetic resonance hydrogen spectrum of the cationic polyrotaxane CPR (0) synthesized by this method ( 1 H NMR, D2O, 298K) Figure 2 As shown, the characteristic peaks of cationic ligands at 4.4ppm and 3.25ppm confirm the successful modification of cationic ligands. The average number of ring penetrations of cationic polyrotaxane is 105, the ring penetration rate is 26.5%, and the average number of cationic ligands per ring is 4.9.

[0052] Example 2 Synthesis of Cationic Ring-Locked Polyrotaxane

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

[0054] (3) Synthesis of Ring-locked Polyrotaxane

[0055] The blocked polyrotaxane was dissolved in a 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, and 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 dialyzate was centrifuged, the white precipitate product was separated, and freeze-dried to obtain the polyrotaxane after the ring lock, which was recorded as PR (1.1).

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

[0057] Take the above-mentioned cyclolocked polyrotaxane (51 mg, 0.0003664 mmol) and dissolve it in 1 mL of 1 mol / L sodium hydroxide solution, and stir until it is completely dissolved. Then dissolve 2,3-epoxypropyltrimethylammonium chloride (809 mg, 5.3344 mmol) in 1 mL of 1 mol / L sodium hydroxide solution, and slowly add the solution dropwise to the sodium hydroxide solution containing the cyclolocked polyrotaxane, and react at room temperature overnight. After the reaction is completed, the product is dialyzed with pure water until the dialyzate is neutral. The dialyzed product is freeze-dried to obtain a crude product. The freeze-dried crude product is washed with an appropriate amount of anhydrous dichloromethane, stirred at room temperature for 24 hours, separated by suction filtration to obtain a white solid product, and vacuum dried at room temperature for 24 hours to obtain the final product, which is recorded as CPR (1.1), that is, the average number of bridging bonds per ring molecule is 1.1.

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

[0059] Example 3 Synthesis of Cationic Ring-Locked Polyrotaxane

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

[0061] (3) Synthesis of Ring-locked Polyrotaxane

[0062] The blocked polyrotaxane was dissolved in a 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, and 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 dialyzate was centrifuged, the white precipitate product was separated, and lyophilized to obtain the polyrotaxane after the ring lock, which was recorded as PR (2.1), that is, the average number of bridging bonds per ring molecule was 2.1.

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

[0064] Take the above-mentioned cyclolocked polyrotaxane (52.6 mg, 0.0003664 mmol) and dissolve it in 1 mL of 1 mol / L sodium hydroxide solution, and stir until it is completely dissolved. Then dissolve 2,3-epoxypropyltrimethylammonium chloride (789 mg, 5.2056 mmol) in 1 mL of 1 mol / L sodium hydroxide solution, and slowly add the solution dropwise to the sodium hydroxide solution containing the cyclolocked polyrotaxane, and react overnight at room temperature. After the reaction is completed, the product is dialyzed with pure water until the dialyzate is neutral. The dialyzed product is freeze-dried to obtain a crude product. The freeze-dried crude product is washed with an appropriate amount of anhydrous dichloromethane, stirred at room temperature for 24 hours, and separated by suction filtration to obtain a white solid product, which is vacuum dried at room temperature for 24 hours to obtain the final product, recorded as CPR (2.1).

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

[0066] Example 4 Synthesis of Cationic Ring-Locked Polyrotaxane

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

[0068] (3) Synthesis of Ring-locked Polyrotaxane

[0069] The blocked 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, and 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 crude product was freeze-dried to obtain a crude product. The freeze-dried crude product was washed with an appropriate amount of anhydrous dichloromethane, stirred at room temperature for 24 hours, and a white solid product was separated by suction filtration. The product was vacuum dried at room temperature for 24 hours to obtain a polyrotaxane after ring locking, which was recorded as PR (3.8), that is, the average number of bridging bonds per ring molecule was 3.8.

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

[0071] Take the above-mentioned cyclolocked polyrotaxane (54.6 mg, 0.0003664 mmol) and dissolve it in 1 mL of 1 mol / L sodium hydroxide solution, and stir until it is completely dissolved. Then dissolve 2,3-epoxypropyltrimethylammonium chloride (755 mg, 4.98 mmol) in 1 mL of 1 mol / L sodium hydroxide solution, and slowly add the solution dropwise to the sodium hydroxide solution containing the cyclolocked polyrotaxane, and react overnight at room temperature. After the reaction is completed, the product is dialyzed with pure water until the dialyzate is neutral. The dialyzed product is freeze-dried to obtain a crude product. The freeze-dried crude product is washed with an appropriate amount of anhydrous dichloromethane, stirred at room temperature for 24 hours, separated by suction filtration to obtain a white solid product, and vacuum dried at room temperature for 24 hours to obtain the final product, recorded as CPR (3.8).

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

[0073] Example 5 Synthesis of Cationic Ring-Locked Polyrotaxane

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

[0075] (3) Synthesis of Ring-locked Polyrotaxane

[0076] The blocked 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, and 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 crude product was freeze-dried to obtain a crude product. The freeze-dried crude product was washed with an appropriate amount of anhydrous dichloromethane, stirred at room temperature for 24 hours, and a white solid product was separated by suction filtration. The product was vacuum dried at room temperature for 24 hours to obtain a polyrotaxane after ring locking, which was recorded as PR (5.9), that is, the average number of bridging bonds per ring molecule was 5.9.

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

[0078] Take the above-mentioned cyclolocked polyrotaxane (57.4 mg, 0.0003664 mmol) and dissolve it in 1 mL of 1 mol / L sodium hydroxide solution, and stir until it is completely dissolved. Then dissolve 2,3-epoxypropyltrimethylammonium chloride (710 mg, 4.68 mmol) in 1 mL of 1 mol / L sodium hydroxide solution, and slowly add the solution dropwise to the sodium hydroxide solution containing the cyclolocked polyrotaxane, and react at room temperature overnight. After the reaction is completed, the product is dialyzed with pure water until the dialyzate is neutral. The dialyzed product is freeze-dried to obtain a crude product. The freeze-dried crude product is washed with an appropriate amount of anhydrous dichloromethane, stirred at room temperature for 24 hours, separated by suction filtration to obtain a white solid product, and vacuum dried at room temperature for 24 hours to obtain the final product, recorded as CPR (5.9).

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

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

[0081] The Zeta potential data of the cationic polyrotaxanes prepared in Examples 1-5 are as follows: Figure 7 As shown in the figure, it can be clearly seen that even if the average number of cationic ligands per ring of each group of polyrotaxane samples is the same, 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 the axis, ring and ligand of the polyrotaxane is the same, the spatial arrangement of the ring molecules is restricted by the bridging structure, and the bridging structure connects the originally relatively independent ring molecules together through covalent bonds, making the ring molecules more closely arranged, resulting in a more concentrated distribution of cationic ligands on the ring and an increase in the cation density. In the unbridged polyrotaxane, each ring molecule remains relatively independent, and the distribution of cationic ligands will be more dispersed, which makes the cation density carried on each ring molecule lower, resulting in a lower zeta potential. Therefore, as the number of bridging bonds increases, the Zeta potential will be higher.

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

[0083] In order to study the effect of the degree of ring locking of polyrotaxane on its antibacterial properties, cationized polyrotaxanes with different degrees of ring locking were prepared into solutions with the same cationic ligand concentration (0.25 μg / mL) and co-cultured with Escherichia coli at 37°C and 125 rpm for 4 hours. After the culture, the bacterial activity was calculated by the bacterial plate counting method, bacterial activity (%) = number of bacteria in the experimental group / number of bacteria in the control group * 100%. After 4 hours of co-culture, the lower the bacterial activity, the stronger the antibacterial effect of the material.

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

[0085] Example 7 Hemolysis Experiment of Cationic Ring-Locked Polyrotaxane

[0086] In order to evaluate the blood compatibility of the cationic polyrotaxanes prepared in Examples 1-5, the hemolysis rate was used to characterize the effect of the materials on red blood cells. The hemolysis rate refers to the proportion of red blood cells that dissolve after contacting the material. If the hemolysis rate of the material exceeds 5%, it is considered to have a certain degree of toxicity and may cause potential adverse effects on blood or host tissues. Through this experiment, the safety of cationic polyrotaxanes when used in vivo can be preliminarily judged, especially the reaction when in contact with blood.

[0087] Blood compatibility results such as Fig. 9 As shown, each group of cationic polyrotaxanes did not produce obvious hemolysis within the range of cationic ligand concentration of 0 to 64 μg / mL, and the hemolysis rate was less than 5%, which met the blood safety requirements.

[0088] Example 8 Cytotoxicity Experiment of Cationic Ring-Locked Polyrotaxane

[0089] In order to characterize the cytotoxicity of the material, mouse embryonic fibroblasts NIH 3T3 were selected to evaluate the cytotoxicity of the material. The cells were seeded in a 96-well plate, and the culture plate was placed in an incubator for pre-culture (37°C, 5% CO2) for 12-24 hours to allow the cells to reach the exponential phase. The culture medium of each well was aspirated, and the material with a cationic ligand concentration of 1 μg / mL was added to the culture plate for co-culture with the cells for 24 hours. After 24 hours, the cell activity was detected using the CCK-8 kit according to the operating instructions, with fresh culture medium as a control, and 5 parallels per group.

[0090] Cytotoxicity results such as Fig.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 obvious toxicity to the cells and met the biosafety requirements.

[0091] Example 9 Synthesis of Ring-Locked Polyrotaxane

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

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

[0094] The experimental results show that terephthalate can also be used as a bridging agent to adjust the degree of ring locking of the polyrotaxane ring molecule. After modifying the cation, the antibacterial effect of the cationic polyrotaxane is significantly enhanced when the concentration of the cationic ligand is the same.

[0095] Example 10 Synthesis of Cationic Ring-Locked Polyrotaxane

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

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

[0098] The experimental results show that the antibacterial effect of the cationic polyrotaxane modified with pentaethylenehexamine is significantly enhanced when the concentration of cationic ligands is the same.

Claims

1. A cationic ring-locked polyrotaxane, characterized in that: The polyrotaxane comprises an axis molecule, a ring molecule, an end-capping structure, and a connection structure formed by chemical reaction between coaxially adjacent ring molecules and hydroxyl groups on adjacent ring molecules; the axis 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.

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

3. The cationic ring-locked polyrotaxane according to claim 1, characterized in that: The cationic ligands are 2,3-epoxypropyltrimethylammonium chloride and pentaethylenehexamine.

4. The cationic ring-locked polyrotaxane according to claim 1, characterized in that: The axle molecule is polyethylene glycol or polyethylene glycol block copolymer with a molecular weight of 4000 to 35000 Da.

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

6. The cationic ring-locked polyrotaxane according to claim 1, characterized in that: The end-capping structure is formed by the reaction of adamantane amine or 2,4-dinitrofluorobenzene with the terminal carboxyl group of the axle molecule.

7. The method for preparing the cationic ring-locked polyrotaxane according to any one of claims 1 to 6, 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 polyrotaxane; the bridging agent is one of epichlorohydrin or diisocyanate compounds.

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

9. The method according to claim 7, characterized in that: The diisocyanate compounds are terephthalate diisocyanate and o-phthalate diisocyanate.

10. Use of the cationic ring-locked polyrotaxane according to any one of claims 1 to 6 in antibacterial applications or in the preparation of antibacterial products.

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