Cationic polymer antibiotic delivery carrier as well as preparation method and application thereof
By using antibiotic delivery vectors constructed with ε-PLL and cyclodextrin, electrostatic and supramolecular effects to assist in deep penetration and enrichment of antibiotics, the existing vectors have solved the shortcomings of the bacterial biofilm barrier and achieved efficient antibiotic delivery and biofilm removal effects.
Patent Information
- Application Number
- CN202411972763.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-30
- Publication Date
- 2025-05-06
AI Technical Summary
Existing cationic polymer antibiotic delivery vectors have problems such as cytotoxicity, poor biocompatibility and difficulty in deep penetration in overcoming bacterial biofilm barriers, resulting in the gradient distribution of antibiotics in biofilm and increasing bacterial resistance.
The antibiotic delivery vector constructed with ε-polylysine (ε-PLL) and cyclodextrin (CD) achieves deep penetration through the reversible electrostatic action of ε-PLL and biofilm, and conveniently binds antibiotics through the supramolecular action of cyclodextrin and antibiotics to assist antibiotics to overcome the biofilm barrier.
The efficient enrichment and uniform distribution of antibiotics in biofilms has been achieved, which significantly improves the removal ability of antibiotics to bacterial biofilms and reduces the risk of bacterial resistance.
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Figure CN119931022A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedical technology, and in particular to a cationic polymer antibiotic delivery carrier and a preparation method and application thereof. Background Art
[0002] Research data show that 80% of infection cases are related to biofilms formed by bacteria on the surface of tissues or plants. Biofilms are formed by bacteria aggregating and adhering to the interface of tissues or plants and secreting a large amount of extracellular polymeric substance (EPS) including DNA, mucopolysaccharides and proteins. On the one hand, biofilms provide a favorable environment for the survival and proliferation of bacteria. On the other hand, dense, highly negatively charged EPS can effectively hinder the entry of antibacterial elements such as antibiotics and immune cells. The concentration of antibiotics required to eradicate biofilms is usually 100-1000 times the minimum inhibitory concentration of suspended bacteria, but in clinical treatment, it is extremely difficult for antibiotics to reach this concentration at the site of infection to completely eliminate bacteria in biofilms. In addition, the obstruction of antibiotic penetration by biofilms causes antibiotics to be distributed in a gradient, inducing a sublethal state of bacteria, thereby increasing drug resistance. Therefore, once formed, biofilms are extremely difficult to remove, and are a key factor in causing bacterial resistance, chronic and stubborn infections.
[0003] Antibiotics are the core treatment for infectious diseases. How to improve the efficiency of antibiotics in eradicating bacteria in biofilms is the key to successfully treating biofilm-related infections. In recent years, nano-drug carriers combined with antibiotics and other drugs have been widely studied in the removal of biofilm infections, providing more efficient drug treatment options for infection-related diseases. Although traditional nano-delivery carriers such as liposomes, PCL nanospheres, and PLGA nanospheres can improve the antibacterial efficiency of antibiotics to a certain extent, these carrier materials have poor biofilm enrichment, penetration, and retention capabilities, low efficiency in interacting with bacteria, and very limited delivery efficiency, and cannot eradicate biofilm infections and prevent recurrence of infections. Biofilms are composed of a large number of negatively charged matrix polymers (eDNA, mucopolysaccharides, etc.), which are significantly negatively charged under physiological conditions. In recent years, nano-drug delivery systems constructed based on cationic polymers (such as α-polylysine α-PLL, polyethyleneimine PEI, quaternary ammonium salt-modified polymers, etc.) have received widespread attention in the fight against biofilm infections. They can quickly and efficiently enrich, penetrate, and reside in biofilms through electrostatic effects, and can also adsorb and destroy bacterial outer membranes through charge effects to exert antibacterial effects. Therefore, drug delivery systems constructed by cationic polymers can usually overcome the barrier effect of biofilms more efficiently, improve the antibacterial efficiency of antibiotics and the ability to clear biofilm infections. However, the cationic polymers currently used face many problems. First, cationic polymers usually have significant cytotoxicity, poor biocompatibility and no degradation ability; second, the cationic polymers currently used are too highly charged, resulting in limited biofilm penetration ability, making it difficult to diffuse into the deep layers of the biofilm, thereby causing a gradient distribution of drugs in the biofilm, which is not conducive to eliminating dormant bacteria in the lower layer and may aggravate bacterial resistance. Summary of the invention
[0004] The purpose of the present invention is to provide a cationic polymer antibiotic delivery carrier and a preparation method and application thereof in view of the deficiencies in the prior art.
[0005] To achieve the above object, the technical solution adopted by the present invention is:
[0006] The first aspect is to provide a cationic polymer antibiotic delivery carrier, the structure of which is shown in the following formula (I):
[0007]
[0008] Wherein, R is β-cyclodextrin or γ-cyclodextrin; R1 is H, β-cyclodextrin or γ-cyclodextrin; m and n are positive integers, and 100>m+n>10.
[0009] Furthermore, the number of lysine monomers modified by cyclodextrin in the antibiotic delivery carrier accounts for 1% to 50% of the total number of lysine monomers.
[0010] ε-poly-l-lysine (ε-PLL) is a cationic polypeptide containing 25 to 35 lysines secreted by microorganisms, which has broad-spectrum antibacterial properties. ε-PLL is polymerized by the ε-amino group of lysine and the α-carboxyl group, and its protonable group is the α-amino group of lysine. Compared with the primary, secondary, and tertiary amino groups in the ε-amino group and other cationic polymers, the α-amino group has a lower pKa value and weaker protonation ability. Therefore, ε-PLL has a low positive charge density and can undergo reversible electrostatic interaction with negatively charged biofilms, achieving deep penetration and uniform distribution in the biofilm.
[0011] Antibiotic molecules generally do not have functional groups that bond to ε-PLL; in addition, changes in the structure of antibiotics are very likely to affect their antibacterial ability. Cyclodextrin (CD) is a cyclic oligosaccharide with a hydrophilic surface and a hydrophobic inner cavity formed by the action of amylose by specific microorganisms. Its hydrophobic inner cavity can bind to the hydrophobic structure of fat-soluble small molecules or drug molecules through supramolecular action. In the present invention, CD can conveniently realize the non-covalent bond between antibiotics and carriers through supramolecular action of host-guest recognition with antibiotic molecules, and then use the electrostatic action of ε-PLL and biofilm to assist antibiotics in overcoming the barrier effect of bacterial biofilm.
[0012] The second aspect is to provide a method for preparing the above-mentioned cationic polymer antibiotic delivery carrier, comprising the following steps:
[0013] Step 1, accurately weigh cyclodextrin powder, add dimethyl sulfoxide (DMSO) to dissolve; under stirring conditions, add Dess-Martin oxidant to the above solution, stir and react at 20-35° C. for 1-5 hours, and after the reaction is completed, perform post-treatment to obtain aldehyde-modified cyclodextrin (Ald-CD);
[0014] Step 2, accurately weigh ε-PLL hydrochloride powder and fully dissolve it in acetic acid / sodium acetate buffer; add the aldehyde-modified cyclodextrin prepared in step 1 to the above solution under stirring conditions, stir at 40-50° C. for 24-36 hours, add reducing agent sodium cyanoborohydride, stir at room temperature for 3-5 days, after the reaction is completed, put the reaction solution into a dialysis bag, and dialyze with ultrapure water; finally, freeze-dry to obtain ε-PLL-CD powder.
[0015] Furthermore, in the step 1, the mass ratio of dimethyl sulfoxide to cyclodextrin is (10-20):1; and the molar ratio of Dess-Martin oxidant to cyclodextrin is (1-2):1.
[0016] Furthermore, in the step 1, the specific steps of post-treatment are: adding acetone to the solution after the reaction is completed to precipitate, centrifuging the precipitate, and discarding the supernatant;
[0017] Deionized water is added to the precipitate, ultrasonic-assisted dissolution is performed, impurities in the precipitate are removed by filtration, and a filtrate is retained; acetone is added to the filtrate, the precipitate is collected by centrifugation, and the aldehyde-modified cyclodextrin is obtained after drying.
[0018] Furthermore, in the step 2, the amount of aldehyde-modified cyclodextrin added is 0.5 to 2 times the amount of ε-PLL lysine monomer; and the amount of sodium cyanoborohydride used is 6 to 10 times the molar number of the aldehyde-modified cyclodextrin fed.
[0019] The third aspect is to provide a kit comprising the above cationic polymer antibiotic delivery carrier, wherein the kit also comprises pharmaceutical grade antibiotic powder and sterile physiological saline.
[0020] Furthermore, in the kit, the antibiotic used is one of penicillins, cephalosporins, macrolides, oxazolidinones, azithromycin, rifamycins, and glycopeptide antibiotics.
[0021] Furthermore, the method of using the kit is as follows: sterile physiological saline is used to dissolve the antibiotic delivery carrier powder and the pharmaceutical-grade antibiotic powder respectively, followed by mixing, and then locally injecting the mixture into the infected area.
[0022] Furthermore, the concentration of the antibiotic delivery vector in the final mixed solution is 0.5-100 mg / mL, and the mass concentration of the antibiotic is 10-100% of the mass concentration of the antibiotic delivery vector.
[0023] The present invention adopts the above technical solution, and has the following technical effects compared with the prior art:
[0024] The present invention uses ε-PLL and CD, which are widely used in the medical field, to construct a low-cost, topical antibiotic delivery carrier for bacterial biofilm infection. The ε-PLL-CD carrier of the present invention can quickly enrich, penetrate and evenly distribute the biofilm at the infection site through the reversible electrostatic interaction with the negatively charged biofilm, and at the same time, bind the antibiotic molecules through the supramolecular interaction between cyclodextrin and the antibiotic, so as to help the antibiotic molecules to be efficiently enriched at the biofilm infection site, overcome the penetration barrier effect of the biofilm on the antibiotic, and achieve efficient anti-biofilm infection removal efficiency. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Figure 1 The cell compatibility of the ε-PLL-CD prepared in Example 1 was shown, wherein the test cells used were fibroblasts.
[0026] Figure 2 The ability of ε-PLL-CD to load linezolid-enriched biofilms was demonstrated.
[0027] Figure 3 The ability of ε-PLL-CD loaded with linezolid to remove Staphylococcus aureus biofilm on the surface of metal implants was demonstrated.
[0028] Figure 4 The positive rate of infection at the injection site in the animal experiment of Example 6 is shown.
[0029] Figure 5 The positive rate of bone tissue infection at the injection site in the animal experiment of Example 6 is shown.
[0030] Figure 6 The positive rate of joint capsule infection at the injection site in the animal experiment of Example 6 is shown.
[0031] Figure 7 The positive rate of Kirschner wire infection at the injection site in the animal experiment of Example 6 is shown. DETAILED DESCRIPTION
[0032] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments, but is not intended to be limiting of the present invention. It should be noted that the embodiments and features in the embodiments of the present invention may be combined with each other without conflict.
[0033] Example 1 Preparation of ε-PLL-CD (β-cyclodextrin), cytotoxicity evaluation and biofilm enrichment ability test
[0034] Accurately weigh 1.13 g (1 mmol) of β-cyclodextrin, dissolve it in 10 mL DMSO with ultrasound assistance; add 0.848 g (2 mmol) of Dess-Martin oxidant to the above solution under stirring, and react at 25°C for 1 hour. Subsequently, add the reaction solution to 200 mL of acetone, centrifuge at 4500 rpm for 5 minutes, and discard the supernatant; add 10-20 mL of deionized water to the precipitate, dissolve the precipitate again with ultrasound assistance, centrifuge at 4500 rpm for 5 minutes, and discard the precipitate; add the above centrifuged clear liquid to 200 mL of acetone, collect the precipitate by centrifugation and dry it for 24 hours (37°C) to obtain 0.5 g of aldehyde-modified β-cyclodextrin (Ald-β-CD).
[0035] Accurately weigh 0.2g of ε-PLL hydrochloride, dissolve it in 5mL acetic acid / sodium acetate buffer (pH=7.2, 0.1M), and stir until completely dissolved. Under stirring conditions, add 0.5g (0.44mmol) of the product (Ald-β-CD) prepared above to the solution and stir at 40°C for 24 hours. Subsequently, 0.17g of sodium cyanoborohydride (2.7mmol) was added to the reaction system at room temperature and stirring was continued for 3 days. After the reaction was completed, unreacted impurities and foreign ions were removed by dialysis (3.5KDa molecular weight cutoff), and the dialysis time was 4d, during which the dialysis fluid was replaced several times. The dialyzed product was freeze-dried to obtain 0.35g of white powder, i.e., ε-PLL-CD. The results of characterization by hydrogen nuclear magnetic resonance spectroscopy showed that the modification rate of CD in ε-PLL-CD was 20.47%.
[0036] The cell viability staining technique was used to evaluate the cytotoxicity of the prepared ε-PLL-CD. The experimental results showed that 1 mg / mL ε-PLL-CD did not show significant cytotoxicity to fibroblasts (see Figure 1 ).
[0037] Example 2 Antibacterial kit composed of ε-PLL-CD and linezolid
[0038] 10 mg of the ε-PLL-CD powder and 1 mg of linezolid powder prepared in Example 1 were dissolved in deionized water, and sterilized with a 220 nm filter membrane. Subsequently, the above solutions were loaded into 2.5 mL cillin bottles, freeze-dried and packaged under aseptic conditions, and formed into an antibacterial kit with sterile saline. Subsequently, 0.5 mL of sterile saline was taken to fully dissolve the ε-PLL-CD and linezolid powders, and the two were evenly mixed, and ultrasound-assisted mixing was performed for 5 minutes. The resulting mixed solution of the carrier (ε-PLL-CD) combined with the antibiotic molecule was used for subsequent antibacterial testing.
[0039] Example 3 The ability of ε-PLL-CD to carry linezolid to enrich biofilm
[0040] Staphylococcus aureus was planted in culture dishes and cultured for a certain period of time to form a biofilm. Subsequently, 10 mg / L carrier-loaded linezolid or 10 mg / mL linezolid without a carrier was added to the culture dishes forming the biofilm. After 6 hours of culture, the culture solution was collected and filtered through a 220 nm filter membrane. The residual linezolid content in the culture medium was detected by a UV-visible spectrophotometer. Experimental results ( Figure 2 ) showed that the residual amount of antibiotics in the culture medium after 6 hours in the carrier-loaded linezolid group was significantly less than that in the simple antibiotic group. This result proves that the carrier can effectively help antibiotics enrich in biofilms.
[0041] Example 4 Antibacterial ability test of linezolid combined with carrier
[0042] To determine the minimum inhibitory concentration (MIC) of linezolid bound to the carrier, a suspension of Staphylococcus aureus was first prepared, its concentration was adjusted to the 0.5 McFarland standard, and further diluted to 1-5 × 10 5 CFU / mL. Using Mueller-Hinton broth (MHB) as the dilution medium, the linezolid containing the carrier prepared in Example 2 was diluted with a concentration gradient from 256 μg / mL to 0.25 μg / mL, and 100 μL of the dilution was added to each well of the 96-well plate. Subsequently, 100 μL of bacterial suspension was added to each well, with a total volume of 200 μL, and positive and negative controls were set up. After the 96-well plate was placed in a 37°C incubator and cultured for 18-24 hours, the bacterial growth was read. The results were read by a spectrophotometer, and the MIC value was defined as the lowest drug concentration at which no visible bacterial growth was observed in the well.
[0043] The experimental results showed that the MIC of linezolid and linezolid bound to the carrier were both 2 mg / L. This result showed that the carrier molecule did not affect the antibacterial properties of the antibiotic.
[0044] Example 5 Test on the ability of linezolid combined with carrier to eliminate bacterial biofilm in vitro
[0045] The present invention uses a titanium alloy disc to simulate the surface of a metal implant in bone tissue. After Staphylococcus aureus is plated, it is statically cultured at 37°C for 24 hours, and a single colony is picked and subcultured into 5 mL of TSB culture medium and incubated on a shaker at 37°C for 16 to 24 hours to obtain the original bacterial solution. The test is divided into groups: a blank control group (Control group) (only TSB culture medium, original bacterial solution, and titanium alloy disc are added); an ε-PLL-CD group (TSB culture medium, original bacterial solution, titanium alloy disc, and ε-PLL-CD solution are added); a linezolid (LZD) group (TSB culture medium, original bacterial solution, titanium alloy disc, and LZD solution are added); an ε-PLL-CD (ε-PLL-CD-LZD) group loaded with LZD (TSB culture medium, original bacterial solution, titanium alloy disc, and ε-PLL-CD-LZD solution diluted from the mixed solution in Example 2 are added).
[0046] Place a titanium alloy disc with a diameter of about 1 cm in a 24-well plate, then add about 2 mL of TSB medium, and then add 20 uL of the original bacterial solution and culture at 37°C for 48 hours. Carefully remove the titanium alloy disc and place it in a new 24-well plate, then take another 1.8 mL of TSB medium, add 20 uL of ε-PLL-CD solution, LZD solution, and ε-PLL-CD-LZD solution to each well and mix by pipetting. Subsequently, 2 mL of a mixed solution of TSB + ε-PLL-CD (carrier concentration 20 mg / L) was added to the ε-PLL-CD group, 2 mL of a mixed solution of TSB + LZD (LZD concentration 2 mg / L) was added to the LZD group, 2 mL of a mixed solution of TSB + ε-PLL-CD-LZD (carrier concentration 20 mg / L, antibiotic concentration 2 mg / L) was added to the ε-PLL-CD-LZD group, and 2 mL of TSB culture medium was added to the blank control group, and the plates were cultured at 37°C for 24 h. Subsequently, the titanium alloy discs were taken out and fixed for scanning electron microscopy.
[0047] The experimental results are as attached Figure 3 As shown, the number of biofilm and bacteria in the ε-PLL-CD-LZD group was significantly less than that in the other groups and was obviously destroyed, the number of biofilm and bacteria in the ε-PLL-CD and LZD groups was significantly less than that in the blank control group and was destroyed, the number of biofilm and bacteria in the LZD group was slightly less than that in the ε-PLL-CD group and the degree of destruction was higher than that in the ε-PLL-CD group, and obvious biofilm and a large number of Staphylococcus aureus were observed in the blank control group.
[0048] Example 6 Test on the effect of linezolid combined with carrier on bone and joint infection in SD rats
[0049] The present invention uses the SD rat joint implant infection model to test the ability of ε-PLL-CD loaded with linezolid to resist bone and joint infection. First, select adult male SD rats (250-300g) and make a skin incision on the outside of the rat thigh. The incision is about 1.5-2cm, and the skin and muscle tissue are cut layer by layer until the middle part of the femur is exposed. Use blunt separation method to separate the muscle tissue, completely expose the diaphysis of the femur, and then use a micro-electric drill to make a transverse fracture line in the middle part of the femur. Use a micro-electric drill to drill a small hole in the femoral condyle to insert the internal fixation needle. A sterile surgical grade Kirschner wire (diameter 0.088 mm; length 20 mm) is inserted from the distal condyle of the femur to the proximal end of the femur, ensuring that the needle passes completely through the fracture site and the protruding part is placed in the knee joint. The incision is then sutured, and Staphylococcus aureus (the amount of bacteria inoculated is about 10 6 -10 8CFU / mL) was injected into the arthrotomy site. The incision was kept closed for 7 days to allow bacteria to attach to the implant and adjacent tissues. After 7 days, 100uL PBS solution, 100uL linezolid (30mg / kg body weight) solution, 100uL ε-PLL-CD solution (300mg / kg body weight), and 100uL LZN (30mg / kg body weight, diluted by the mixed solution of the kit of the specific embodiment 2) solution loaded by ε-PLL-CD (300mg / kg body weight) were injected into the rat knee arthrotomy site every day, and the intervention lasted for 7 days. Finally, the Levine method was used to guide the swab sampling and culture of the implant and the exposed tissue (Kirschner wire, bone and joint capsule) with a cotton swab, and the infection positive rate of the above-mentioned parts was determined.
[0050] The test results are as follows Figure 4-Figure 7 As shown. In the chi-square test, the differences in the positive rates of each group in all parts were statistically significant (P < 0.05). The experimental results show that the LNZ group loaded with the ε-PLL-CD carrier performed best in controlling infection, and the culture positivity rates of the joint as a whole, Kirschner wire, bone, and joint capsule were significantly lower than those of other groups. The above experimental results prove that the ε-PLL-CD carrier can significantly improve the ability of antibiotics to clear bacteria. The test of specific embodiment 2 shows that the MIC of LNZ loaded with ε-PLL-CD is the same as that of LNZ without a carrier against bacteria, and the in vivo test results show that the carrier can improve the anti-infection ability of antibiotics. The reason is that the carrier can more efficiently assist antibiotics in enriching biofilm sites in actual infection treatment.
[0051] The above description is only a preferred embodiment of the present invention, and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the contents of the specification and illustrations of the present invention should be included in the protection scope of the present invention.
Claims
1. A cationic polymer antibiotic delivery carrier, characterized in that: Its structure is shown in the following formula (I): Wherein, R is β-cyclodextrin or γ-cyclodextrin; R1 is H, β-cyclodextrin or γ-cyclodextrin; m and n are positive integers, and 100>m+n>10.
2. The cationic polymer antibiotic delivery carrier according to claim 1, characterized in that: The number of lysine monomers modified by cyclodextrin in the antibiotic delivery carrier accounts for 1% to 50% of the total number of lysine monomers.
3. A method for preparing a cationic polymer antibiotic delivery carrier as described in any one of claims 1 to 2, characterized in that: The steps include: Step 1, accurately weigh cyclodextrin powder, add dimethyl sulfoxide to dissolve; add Dess-Martin oxidant to the above solution under stirring conditions, stir and react at 20-35° C. for 1-5 hours, and after the reaction is completed, perform post-treatment to obtain aldehyde-modified cyclodextrin; Step 2, accurately weigh ε-PLL hydrochloride powder and fully dissolve it in acetic acid / sodium acetate buffer; add the aldehyde-modified cyclodextrin prepared in step 1 to the above solution under stirring conditions, stir at 40-50° C. for 24-36 hours, add reducing agent sodium cyanoborohydride, stir at room temperature for 3-5 days, after the reaction is completed, put the reaction solution into a dialysis bag, and dialyze with ultrapure water; finally, freeze-dry to obtain ε-PLL-CD powder.
4. The preparation method according to claim 3, characterized in that: In the step 1, the mass ratio of dimethyl sulfoxide to cyclodextrin is (10-20):1; the molar ratio of Dess-Martin oxidant to cyclodextrin is (1-2):
1.
5. The preparation method according to claim 3, characterized in that: In the step 1, the specific steps of post-treatment are: adding acetone to the solution after the reaction is completed to precipitate, centrifuging the precipitate, and discarding the supernatant; adding deionized water to the precipitate, ultrasonically dissolving it, filtering to remove impurities in the precipitate, and retaining the filtrate; adding acetone to the filtrate, centrifuging to collect the precipitate, and drying to obtain the aldehyde-modified cyclodextrin.
6. The preparation method according to claim 3, characterized in that: In the step 2, the amount of the aldehyde-modified cyclodextrin added is 0.5 to 2 times the amount of the ε-PLL lysine monomer substance; the amount of sodium cyanoborohydride used is 6 to 10 times the molar number of the aldehyde-modified cyclodextrin fed.
7. A kit comprising the cationic polymer antibiotic delivery carrier according to any one of claims 1 to 2, characterized in that: The kit also includes pharmaceutical grade antibiotic powder and sterile physiological saline.
8. The kit according to claim 7, characterized in that In the kit, the antibiotic used is one of penicillins, cephalosporins, macrolides, oxazolidinones, azithromycin, rifamycins, and glycopeptide antibiotics.
9. The kit according to claim 7, characterized in that The method for using the kit is as follows: sterile physiological saline is used to dissolve the antibiotic delivery carrier powder and the pharmaceutical-grade antibiotic powder respectively, and then the two are mixed.
10. The kit according to claim 9, characterized in that The concentration of the antibiotic delivery vector in the final mixed solution is 0.5-100 mg / mL, and the mass concentration of the antibiotic is 10-100% of the mass concentration of the antibiotic delivery vector.