Polymer and application thereof
By designing ROS-sensitive polymers and polymerizing them with monomer A to form nanoparticles, the problem of low efficiency of existing antioxidants is solved, achieving efficient ROS scavenging and retinal ganglion cell rescue, with better biosafety and stability.
Patent Information
- Application Number
- CN202410593419.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-05-14
- Publication Date
- 2025-11-14
AI Technical Summary
Existing antioxidant molecules are inefficient and unstable in scavenging excess reactive oxygen species (ROS) in the body, making them ineffective in combating oxidative stress, especially in maintaining cellular health.
A polymer composed of a monomer containing a ROS-sensitive group and monomer A was designed to form nanoparticles through self-assembly, which can be used to remove excess ROS. The polymer is composed of anhydride and diisocyanate units, has high sensitivity and degradability, and can remove excess ROS in vivo, thus saving optic ganglion cells.
This polymer is highly sensitive to ROS and can effectively remove excess ROS, resulting in better therapeutic efficiency and biosafety, extended drug circulation time, and improved stability through nanoparticle form, significantly rescuing optic ganglion cells.
Smart Images
Figure CN120944053A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biochemical technology, specifically to a polymer and its applications, and more particularly to a method for synthesizing ROS-sensitive polymers and preparing nanoparticles, as well as their applications. Background Technology
[0002] Reactive oxygen species (ROS) refer to the collective term for oxygen-containing free radicals and peroxides that readily form free radicals, all of which are involved in oxygen metabolism within organisms. They primarily describe single-electron reducing agents of oxygen in the body, including hydrogen peroxide, superoxide anions, singlet oxygen, and hydroxyl radicals. In a biological context, ROS formation is a natural byproduct of normal oxygen metabolism and plays a crucial role in cell signaling and homeostasis. ROS-induced oxidative stress refers to an imbalance between oxidation and antioxidation in the body, and is an important indicator of aging and disease. Under oxidative stress conditions, excessive ROS damage cellular proteins, lipids, and DNA, leading to fatal cellular damage and consequently affecting various pathologies such as aging, cancer, neurodegenerative diseases, cardiovascular diseases, and diabetes.
[0003] When the anti-ROS system is disrupted, strategies to scavenge ROS through antioxidant molecules (vitamins, carotenoids, and flavonoids) or natural enzymes (superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx)) have broad applicability. While promising for reducing ROS levels, many traditional antioxidant molecules are less efficient than natural enzymes. Furthermore, natural enzymes face several limitations, such as sensitivity to environmental conditions, limited functional stability, and difficulties in large-scale production.
[0004] Besides natural enzymes or metal-based biocatalytic nanostructures, in recent decades, researchers have increasingly focused on exploring novel, highly efficient antioxidant organic molecules with high biocompatibility and degradability. Polymer antioxidants can be introduced into polymeric materials through monomer introduction or post-grafting methods and play a crucial role in maintaining the redox balance in the human body. Antioxidant organic molecules may enhance the reactive oxygen species scavenging capacity of biocatalytic nanostructures through synergistic antioxidant effects. Summary of the Invention
[0005] The purpose of this invention is to provide a polymer and its application, which is highly sensitive to ROS, can remove excessive ROS generated in the body due to the imbalance of the redox system, can resist oxidative stress, and can rescue optic ganglion cells.
[0006] To this end, the present invention provides the following technical solution.
[0007] A first aspect of the present invention provides a polymer polymerized from a monomer containing a ROS-sensitive group and monomer A, wherein monomer A comprises one of an acid anhydride and a diisocyanate, and the polymer has a structure of general formula I or general formula II:
[0008]
[0009] Where X represents a diisocyanate unit, Y represents an anhydride unit, Z represents a monomer unit containing a ROS-sensitive group, and m and n are each independent positive integers.
[0010] The monomer containing ROS-sensitive groups refers to a monomer that can consume ROS.
[0011] Furthermore, the monomer containing the ROS-sensitive group includes a dihydroxy monomer containing the ROS-sensitive group.
[0012] Furthermore, the molar ratio of the monomer containing the ROS-sensitive group to monomer A is 1:1 to 1.5.
[0013] Furthermore, the monomer containing the ROS-sensitive group includes one or more of the following compounds:
[0014]
[0015] Furthermore, the diisocyanate comprises one or more of the following compounds:
[0016]
[0017] Furthermore, the acid anhydride includes one or more of the following compounds:
[0018]
[0019] Furthermore, the monomer containing the ROS-sensitive group includes at least one of compounds 1 to 12, and the monomer A is compound 13.
[0020] Furthermore, the monomer containing the ROS-sensitive group includes at least one of the compounds 1 to 12 described above, and the monomer A is compound 14.
[0021] Furthermore, the monomer containing the ROS-sensitive group includes at least one of the compounds 1 to 12 mentioned above, and the monomer A is compound 15.
[0022] Furthermore, the monomer containing the ROS-sensitive group includes at least one of compounds 1 to 12, and the monomer A is compound 16.
[0023] Furthermore, the monomer containing the ROS-sensitive group includes at least one of compounds 1 to 12, wherein monomer A is compound 17.
[0024] Furthermore, the monomer containing the ROS-sensitive group includes at least one of the compounds 1 to 12 mentioned above, and the monomer A is compound 18.
[0025] Furthermore, the monomer containing the ROS-sensitive group includes at least one of compounds 1 to 12, wherein monomer A is compound 19.
[0026] Furthermore, the monomer containing the ROS-sensitive group includes at least one of the compounds 1 to 12 described above, and the monomer A is compound 20.
[0027] Further, the monomer containing the ROS-sensitive group includes at least one of compounds 1 to 12, and monomer A is compound 21. Further, the polymer includes:
[0028]
[0029]
[0030]
[0031]
[0032]
[0033]
[0034]
[0035]
[0036]
[0037]
[0038] Furthermore, n≥100, for example 100~200, 100~500, 200~300, etc., preferably n≥113.
[0039] Furthermore, the polymer has a molecular weight of 8,000 or more, preferably 14,000 or more.
[0040] A second aspect of the present invention provides a method for preparing the polymer of the first aspect, comprising:
[0041] The monomers containing ROS-sensitive groups and monomer A are dissolved in an organic solvent, and then polymerized at 30–150°C for 12–24 h. The polymer is then capped with polyethylene glycol (preferably polyethylene glycol with only one hydroxyl end, such as mPEG5000-OH) for 12–24 h. After the reaction is complete, the reaction system is cooled to room temperature and dialyzed for 24–48 h using a dialysis bag (the type of dialysis bag depends on the target molecular weight, for example, a dialysis bag with a molecular weight of 8000–14000), changing the water 1–10 times. After dialysis, the system can be freeze-dried under vacuum to obtain the polymer.
[0042] The polymers described above can be made into particles of different sizes as needed, such as nanoparticles.
[0043] A third aspect of the present invention provides a method for preparing nanoparticles of the polymer of the first aspect, comprising:
[0044] Weigh out the polymer from the first aspect and dissolve it in an organic solvent. Add it dropwise to rapidly stirred deionized water, or push it into ultrasonically heated deionized water in one go to self-assemble into nanoparticles. Finally, place the system in a dialysis bag for dialysis for 24-48 hours, changing the water 1-10 times during the process.
[0045] A fourth aspect of the invention provides the use of the polymer of the first aspect or the nanoparticles of the third aspect in anti-oxidative stress and rescue of retinal ganglion cells, for example, for the preparation of a medicament for anti-oxidative stress and rescue of retinal ganglion cells.
[0046] In summary, compared with the prior art, the technical solution of the present invention has the following advantages:
[0047] (1) The polymer provided by the present invention is biodegradable in vivo.
[0048] (2) The polymer provided by the present invention is highly sensitive to ROS, can remove excessive ROS generated in the body due to the imbalance of the redox system, can resist oxidative stress, rescue optic ganglion cells, and has better therapeutic efficiency and higher biosafety than existing clinical drugs.
[0049] (3) The polymer provided by the present invention can self-assemble into nanoparticles, which increases the stability of the drug and prolongs the cycle time.
[0050] (4) The preparation method provided by the present invention has the advantages of simple process flow and high product stability. Attached Figure Description
[0051] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. In the drawings:
[0052] Figure 1 The 1H NMR spectrum of polymer I-1 prepared for Example 1 (DMSO d6, 400MHz);
[0053] Figure 2 Example 1 shows the in vitro antioxidant activity assay of polymer I-1.
[0054] Figure 3 The particle size and distribution diagram of the nanoparticles formed by polymer formula I-1 prepared in Example 1;
[0055] Figure 4 Charge distribution map of the nanoparticles formed by polymer formula I-1 in Example 1;
[0056] Figure 5 Electron micrograph of the nanoparticles formed by polymer formula I-1 prepared in Example 1;
[0057] Figure 6 Cell imaging image of nanoparticles formed by polymer formula I-1 as shown in Example 1;
[0058] Figure 7 Absorption spectroscopy of the in vitro antioxidant activity of the nanoparticles formed by polymer formula I-1 prepared in Example 1;
[0059] Figure 8 Fluorescence image of the in vivo antioxidant activity of nanoparticles formed by polymer formula I-1 in Example 1;
[0060] Figure 9 The image shows the effect of nanoparticles formed by polymer formula I-1 in Example 1 on rescuing retinal ganglion cells. Detailed Implementation
[0061] Exemplary embodiments of the present disclosure will now be described in more detail with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.
[0062] Example 1: Formula I-1 copolymer
[0063]
[0064] Weigh 100 mg (0.6 mol) of 3,5-dithia-1,7-heptanediol and 147 mg (0.65 mol) of lysine diisocyanate (LDI) and add them to a 100 mL round-bottom flask. Add 50 mL of DMF and sonicate to dissolve. Heat to 50 °C and react for 24 h. Then maintain the system at 50 °C, add mPEG5000-OH, and continue the reaction for another 24 h. After cooling the reaction system, transfer the liquid to a dialysis bag with a molecular weight cutoff of 8000 and dialyze for 72 h, changing the distilled water four times during this period. Then, freeze-dry the liquid in the dialysis bag under vacuum to obtain the polymer shown in Formula I-1. The 1H-NMR spectrum (DMSO-d6, 400 MHz) of the polymer shown in Formula I-1 is attached. Figure 1 As shown.
[0065] Example 2: In vitro antioxidant activity detection of formula I-1
[0066] Using a hydrogen peroxide detection kit, 10 mg of polymer I-1 was weighed and dissolved in 1 ml of PBS. Standard H2O2 solution was then added to the solution to achieve a final H2O2 concentration of 1 mM. The mixture was incubated at room temperature for 0.5 h, 2 h, 4 h, and 5 h. After incubation, 50 μL of sample and 100 μL of H2O2 detection reagent were transferred to a 96-well plate and incubated at room temperature for 30 minutes. The absorbance at 560 nm was measured using a microplate reader to determine the remaining H2O2 concentration. The H2O2 scavenging ability of polymer I-1 was calculated, and the results are shown in the attached figure. Figure 2 As shown.
[0067] The results showed that H2O2 consumption increased significantly with prolonged incubation time. Specifically, the percentage of H2O2 consumed was close to 20% at 0.5 h and exceeded 90% at 5 h.
[0068] Example 3: Preparation of Nanoparticles
[0069] Weigh 10 mg of polymer I-1 and 10 mg of mPEG DSPE and dissolve them together in 1 ml of DMSO. Dissolve completely by sonication or heating. Then, add the solution dropwise to 10 ml of vigorously stirred deionized water. After 10 minutes, use a dialysis bag with a molecular weight cutoff of 8000 for dialysis for 24 hours, changing the water three times during this period. Filter the dialysate through a 220 μm filter membrane to prepare the nanoparticle system.
[0070] The size, particle size distribution, and charge distribution of the nanoparticles were characterized using a Malvern Zetasizer Nano ZS90 laser particle size analyzer (Nano ZS, UK). The average particle size (z-average) was 58.94 nm, the polymer dispersion index (PDI) was 0.22, and the zeta potential was -0.2284. The results are shown in the attached figure. Figure 3 , 4 As shown.
[0071] Electron microscopy of nanoparticles: 10 μL of nanoparticle liquid was pipetted onto a copper grid of a 300-mesh ultrathin carbon support film in an electron microscope and allowed to air dry. The morphology of the nanoparticles was then observed using a field emission transmission electron microscope (JEOL JEM-F200). The results are shown in the attached figure. Figure 5 As shown.
[0072] The results showed that the nanoparticles were approximately 60 nm in size, had excellent uniformity, were negatively charged, and electron microscopy revealed that they were spherical in shape.
[0073] Example 4: Fluorescence effect of nanoparticles prepared from polymer formula I-1 in cells.
[0074] Place the plasmid at the bottom of each well of a 24-well plate, and add 1 mL of plasmid containing approximately 1×10⁻⁶ ppm to each well. 6 A suspension of R28 cells was prepared and incubated at 37°C for 24 h. Then, the cells were treated with the nanoparticles for 1 h, 4 h, and 7 h. After washing with PBS, the cells were fixed with paraformaldehyde. Cell nuclei were stained with DAPI, and the cytoskeleton was stained with Alexa Fluor 488. Subsequently, the uptake of the nanoparticles by the cells was qualitatively observed using a two-zone confocal laser scanning microscope. The results are attached. Figure 6 As shown in the image. Red fluorescence represents nanoparticles.
[0075] The results showed that red fluorescence was visible in R28 cells, and the red fluorescence was mainly distributed in the cytoplasm. The fluorescence intensity increased with time, proving that the nanoparticles were taken up by the cells in a time-dependent manner.
[0076] Example 5: In vitro antioxidant activity absorption effect of nanoparticles prepared from polymer of formula I-1
[0077] Using the total antioxidant capacity assay kit (ABTS method), nanoparticles prepared from polymer of formula 1 (10 μL, 100 μM) were mixed with ABTS working solution (200 μL) and reacted in the dark for 5 min, 30 min, 1.5 h, 5 h, and 16 h, followed by incubation at room temperature for 2–6 min. The absorption peaks from 500 nm to 900 nm were measured using a microplate reader. The results are shown in the attached figure. Figure 7 As shown.
[0078] The results showed that the peak value of the absorption peak gradually decreased with the extension of reaction time, proving that the nanoparticles have antioxidant activity in vitro.
[0079] Example 6: In vivo antioxidant fluorescence effect of polymer I-1 used to prepare nanoparticles
[0080] Place the plasmid at the bottom of each well of a 24-well plate, and add 1 mL of plasmid containing approximately 1×10⁻⁶ ppm to each well. 6 R28 cell suspensions were incubated at 37°C for 24 hours. Then, the cells were divided into three groups: one group (Glu group) had its culture medium replaced with 15 mM L-glutamate; another group (NP group) underwent culture medium replacement and was treated with the aforementioned nanoparticles; and the last group (Normal group) received no treatment. After 24 hours, the supernatant was discarded, and the cells were fixed with paraformaldehyde. Cell nuclei were stained blue with DAPI (4',6-diamidinyl-2-phenylindole), and reactive oxygen species (ROS) were stained green with the DCFH-DA (2,7-dichlorofluorescein diacetate) probe. DCFH-DA is a ROS probe that can be oxidized by intracellular ROS to generate DCF with strong green fluorescence. The stronger the green fluorescence, the higher the intracellular ROS level. Subsequently, the intensity of ROS in different groups was qualitatively observed using a two-zone confocal laser scanning microscope. The results are attached. Figure 8 As shown.
[0081] The results showed that the green fluorescence of the Normal group was the weakest, while that of the Glu group was the strongest. The green fluorescence of the NP group treated with nanoparticles was significantly weaker than that of the Glu group, proving that nanoparticles can consume ROS in vivo and reduce ROS levels.
[0082] Example 7: Effect of Formula I-1 polymer on in vivo rescue of retinal ganglion cells.
[0083] Mice were pre-divided into three groups. One group (NP group) received the nanoparticles two days before sample collection and the model drug NMDA one day before sample collection. The other group (Mice group) received NMDA only one day before sample collection. NMDA The last group received no processing; this group is Mice. Norm On the day of sample collection, the retinas of the three groups of mice were removed, laid flat on glass slides, and incubated with primary antibody Brn3a for 24 hours. After washing away the residual primary antibody, the retinas were incubated with Alexa Fluor 555 secondary antibody for 48 hours, and then washed away again. Retinal ganglion cells were marked in red. After mounting, the number of positive cells on the retina was qualitatively observed using a two-zone confocal laser scanning microscope. The results are attached. Figure 9 .
[0084] The results show: Mice NormThe group with the most retinal ganglion cells, Mice NMDA The NP group had the fewest retinal ganglion cells, proving the model was successfully constructed, and the number of retinal ganglion cells in the NP group was significantly greater than that in the Mice group. NMDA The study demonstrated that nanoparticles have the effect of rescuing ganglion cells in vivo.
[0085] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.
Claims
1. A polymer, characterized in that, The polymer is synthesized from a monomer containing a ROS-sensitive group and monomer A, wherein monomer A includes one of an acid anhydride and a diisocyanate, and the polymer has the structure of general formula I or general formula II as follows: (General Formula I), (General Formula II); Where X represents a diisocyanate unit, Y represents an anhydride unit, Z represents a monomer unit containing a ROS-sensitive group, and m and n are each independent positive integers.
2. The polymer according to claim 1, characterized in that, The monomers containing ROS-sensitive groups include dihydroxyl monomers containing ROS-sensitive groups.
3. The polymer according to claim 1, characterized in that, The molar ratio of the monomer containing the ROS-sensitive group to monomer A is 1:1 to 1.
5.
4. The polymer according to claim 1, characterized in that, The monomer containing the ROS-sensitive group includes one or more of the following compounds:
5. The polymer as claimed in claim 1, characterized in that, The diisocyanate includes one or more of the following compounds:
6. The polymer according to claim 1, characterized in that, The acid anhydrides include one or more of the following compounds:
7. The polymer according to claim 1, characterized in that, The polymer comprises:
8. The polymer as claimed in claim 1, characterized in that, n≥100, preferably n≥113.
9. The polymer according to claim 1, characterized in that, The polymer has a molecular weight of 8,000 or more, preferably 14,000 or more.
10. Use of the polymer according to any one of claims 1-9 in the preparation of a medicament for oxidative stress rescue of retinal ganglion cells.